HIP1 cancer markers

ABSTRACT

The present invention relates to compositions and methods for cancer diagnostics and therapeutics, including but not limited to, HIP1 cancer markers. In particular, the present invention provides compositions and methods of using HIP1 in the diagnosis and treatment of epithelial cancers. The present invention thus provides improved compositions and methods for diagnosing and treating some of the most common cancers (e.g., prostate and colon cancers). The present invention additionally provides drugs active against HIP1 and methods for screening for such drugs.

[0001] This application claims priority to Provisional PatentApplication Serial No. not yet assigned, filed Nov. 15, 2001 withExpress Mail Label EL837033508US.

[0002] This invention was made with government support under Grant Nos.KO8 CA76025-05 and RO1 CA82363-02, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods forcancer diagnostics, including but not limited to, HIP1 cancer markers.In particular, the present invention provides compositions and methodsof using HIP1 in the diagnosis and treatment of epithelial cancers. Thepresent invention further provides methods of screening potentialtherapeutic compounds for HIP1 inhibitory properties.

BACKGROUND OF THE INVENTION

[0004] Most forms of cancer do not have diagnostic screening testsavailable. For the cancers that do have screening tests available, thetests are frequently invasive, expensive, and lack strong diagnosticutility.

[0005] For example, afflicting one out of nine men over age 65, prostatecancer (PCA) is a leading cause of male cancer-related death, secondonly to lung cancer (Abate-Shen and Shen, Genes Dev 14:2410 [2000];Ruijter et al., Endocr Rev, 20:22 [1999]). The American Cancer Societyestimates that about 184,500 American men will be diagnosed withprostate cancer in 2001 and 39,200 will die.

[0006] Prostate cancer is typically diagnosed with a digital rectal examand/or prostate specific antigen (PSA) screening. An elevated PSA levelcan indicate the presence of PCA. PSA is used as a marker for prostatecancer because it is essentially restricted to prostate cells. A healthyprostate will produce a stable amount—typically below 4 nanograms permilliliter, or a PSA reading of “4” or less—whereas cancer cells produceescalating amounts that correspond with the severity of the cancer. Alevel between 4 and 10 may raise a doctor's suspicion that a patient hasprostate cancer, while amounts above 50 may show that the tumor hasspread elsewhere in the body.

[0007] The advent of prostate specific antigen (PSA) screening has ledto earlier detection of PCA and significantly reduced PCA-associatedfatalities. Elevated serum PSA levels, however, are often detected inpatients with non-malignant conditions such as benign prostatichyperplasia (BPH), and provide little information about theaggressiveness of the cancer detected.

[0008] In addition, colon cancer, which is the second most frequentlydiagnosed malignancy in the United States as well as the second mostcommon cause of cancer death, lacks an effective screening assay. Theprognosis of colon cancer is clearly related to the degree ofpenetration of the tumor through the bowel wall and the presence orabsence of nodal involvement, thus making early diagnosis very importantfor long-term survival.

[0009] A fecal occult blood test (FOBT) is a test used to check forhidden blood in the stool. Sometimes cancers or polyps can bleed, andFOBT is used to detect small amounts of bleeding. In addition, screeningtests (such as a rectal examination, proctoscopy, and colonoscopy) maybe done regularly in patients who are at high risk of colon cancer orwho have a positive FOBT result. The proctoscopy examination finds abouthalf of all colon and rectal cancers. After treatment, a blood test (tomeasure amounts of carcinoembryonic antigen or CEA in the blood) andx-rays may be done to screen for recurrence. CEA is a serum glycoproteinfrequently used in the management of patients with colon cancer.However, a review of the use of this tumor marker suggests that CEA isnot a valuable screening test for colorectal cancer due to the largenumbers of false-positive and false-negative reports.

[0010] Thus, development of additional serum and tissue biomarkersspecific to cancer such and prostate and colon are needed to supplementthe currently available screening methods.

SUMMARY OF THE INVENTION

[0011] The present invention relates to compositions and methods forcancer diagnostics, including but not limited to, HIP1 cancer markers.In particular, the present invention provides compositions and methodsof using HIP1 in the diagnosis and treatment of epithelial cancers. Thepresent invention further provides methods of screening potentialtherapeutic compounds for HIP1 inhibitory properties.

[0012] Accordingly, in some embodiments, the present invention providesan antibody that specifically binds to HIP1 but does not specificallybind to the normal epithelium of prostate or colon. In some embodiments,the antibody binds to the cancerous epithelium of colon or prostate butdoes not bind to the normal epithelium of prostate or colon. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the monoclonal antibody has substantially the same properties asantibodies secreted by a hybridoma selected from the group consisting ofthose deposited as ATCC numbers pending. In some embodiments, themonoclonal antibody is secreted by a hybridoma with ATCC deposit numberpending. In other embodiments, the monoclonal antibody is secreted by ahybridoma with ATCC deposit number pending. In still furtherembodiments, the monoclonal antibody specifically binds to HIP1 proteinwith low background binding. In yet other embodiments, the monoclonalantibody binds to human and mouse HIP1.

[0013] The present invention also provides a method for detectingcancer, comprising providing a sample from a subject suspected of havingcancer; and detecting the presence or absence of HIP1 in the sample. Insome embodiments, the presence of HIP1 in said sample is indicative ofcancer in the subject. In some embodiments, the cancer is selected fromthe group consisting of prostate cancer and colon cancer. In someembodiments, the sample is a tumor sample. In other embodiments, thesample is a tissue sample. In some embodiments, the tissue sample isselected from the group consisting of prostate tissue and colon tissue.In other embodiments, the sample is selected from the group consistingof serum, plasma, blood, and urine. In some embodiments, detecting HIP1comprises detecting the presence of HIP1 mRNA. In some such embodiments,detecting the presence of HIP1 mRNA comprises exposing the HIP1 mRNA toa nucleic acid probe complementary to at least a portion of the HIP1mRNA. In some embodiments, detecting the presence of HIP1 mRNA comprisesa detection assay selected from the group consisting of a Northern blot,in situ hybridization, reverse-transcriptase polymerase chain reaction,and microarray analysis. In other embodiments, detecting the presence ofHIP1 comprises detecting the presence of a HIP1 polypeptide. In someembodiments, detecting the presence of a HIP1 polypeptide comprisesexposing the HIP1 polypeptide to an antibody that specifically binds toHIP1 but does not specifically bind to the normal epithelium of prostateor colon and detecting the binding of the antibody to said HIP1polypeptide. In some embodiments, the antibody is a monoclonal antibody.In some embodiments, the monoclonal antibody has substantially the sameproperties as antibodies secreted by a hybridoma selected from the groupconsisting of those deposited as ATCC numbers pending. In someembodiments, the method further comprises the step of providing aprognosis to the subject.

[0014] The present invention additionally provides a method forcharacterizing tissue in a subject, comprising providing a tissue samplefrom a subject, wherein the tissue is selected from the group consistingof colon and prostate tissue; and detecting the presence or absence ofHIP1 in the sample, thereby characterizing the tissue sample. In someembodiments, the tissue is tumor tissue. In other embodiments, thetissue is biopsy tissue. In some embodiments, detecting HIP1 comprisesdetecting the presence of HIP1 mRNA. In some embodiments, detecting thepresence of HIP1 mRNA comprises exposing the HIP1 mRNA to a nucleic acidprobe complementary to at least a portion of the HIP1 mRNA. In someembodiments, detecting the presence of HIP1 mRNA comprises a detectionassay selected from the group consisting of a Northern blot, in situhybridization, reverse-transcriptase polymerase chain reaction, andmicroarray analysis. In other embodiments, detecting the presence ofHIP1 comprises detecting the presence of a HIP1 polypeptide. In someembodiments, detecting the presence of a HIP1 polypeptide comprisesexposing the HIP1 polypeptide to an antibody that specifically binds toHIP1 but does not specifically bind to the normal epithelium of prostateor colon and detecting the binding of the antibody to the HIP1polypeptide. In some embodiments, the antibody is a monoclonal antibody.In some embodiments, the monoclonal antibody has substantially the sameproperties as antibodies secreted by a hybridoma selected from the groupconsisting of those deposited as ATCC numbers pending. In someembodiments, the tissue sample is a post-surgical prostate tumor tissuesample and the method further comprises the step of identifying a riskof prostate specific antigen failure based on detecting the presence orabsence of HIP1. In other embodiments, the tissue sample is prostatetumor tissue and characterizing comprises identifying a stage ofprostate cancer in the prostate tumor tissue. In some embodiments, thestage is selected from the group consisting of high-grade prostaticintraepithelial neoplasia, benign prostatic hyperplasia, prostatecarcinoma, and metastatic prostate carcinoma. In other embodiments, thetissue sample is prostate tumor tissue and the method further comprisesthe step of identifying the risk of the tumor metastasizing based ondetecting the presence of HIP1. In still further embodiments, the tissuesample is post-surgical prostate tumor tissue and the method furthercomprises the step of identifying the risk of the tumor recurring basedon detecting the presence of HIP1.

[0015] The present invention additionally provides a kit forcharacterizing cancer in a subject, comprising a reagent thatspecifically detects the presence of absence of expression of HIP1; andinstructions for using the kit for characterizing cancer in the subject.In some embodiments, the reagent comprises an antibody that specificallybinds to HIP1 but does not specifically bind to the normal epithelium ofprostate or colon. In some embodiments, the antibody is a monoclonalantibody. In some embodiments, the monoclonal antibody has substantiallythe same properties as antibodies secreted by a hybridoma selected fromthe group consisting of those deposited as ATCC numbers pending. Inother embodiments, the reagent comprises a nucleic acid probe thatspecifically binds to a HIP1 mRNA. In some embodiments, the instructionscomprise instructions required by the United States Food and DrugAdministration for use in in vitro diagnostic products.

[0016] The present invention also provides a method of screeningcompounds, comprising providing an epithelial cell sample; and one ormore test compounds; and contacting the epithelial cell sample with thetest compound; and detecting a change in HIP1 expression in theepithelial cell sample in the presence of the test compound relative tothe absence of the test compound. In some embodiments, the epithelialcell sample is selected from the group consisting of prostate cancercells and colon cancer cells. In some embodiments, detecting comprisesdetecting HIP1 mRNA. In other embodiments, detecting comprises detectinga HIP1 polypeptide. In some embodiments, the cell is in vitro. In otherembodiments, the cell is in vivo. In some embodiments, the test compoundcomprises an antisense compound. In other embodiments, the test compoundcomprises a drug. In some embodiments, the drug is an antibody. In otherembodiments, the drug specifically binds to HIP1.

[0017] The present invention provides the use of HIP1 as a marker todistinguish cancer cells from normal epithelial cells. The presentinvention further provides use of HIP1 as a marker to distinguishprostate or colon cancer cells from normal prostate or colon epithelium.The present invention additionally provides the use of an antibody thatspecifically binds to HIP1 to distinguish cancer cells from normalepithelial cells. The present invention also provides the use of anantibody that specifically binds to HIP1 to distinguish prostate orcolon cancer cells from normal prostate or colon epithelium.

[0018] In some embodiments, the present invention provides a method ofscreening compounds, comprising: providing a first cell samplecomprising cells expressing wild-type HIP1; a second cell samplecomprising cells, wherein said cells do not express HIP1; one or moretest compounds; and contacting the first and seconds samples with thetest compound; and detecting a decrease in viability in the first samplerelative to the second sample. In some preferred embodiments, thedecrease in viability is due to programmed cell death. In someembodiments, the first and second cell samples comprise embryonicfibroblast cells. For example, in some embodiments, the first cellsample comprises embryonic fibroblast cells derived from wild-type miceand the second cell sample comprises embryonic fibroblast cells derivedfrom HIP1 knockout mice. In other embodiments, the first and second cellsamples comprise first and second human cancer cell lines. In someembodiments, the second cancer cell line comprises colo205 cells. Insome embodiments, the test compound comprises a library of testcompounds. In some embodiments, the test compound comprises a lipidanalogue. In some embodiments, the test compound binds to HIP1. Forexample, in some embodiments, thetest compound binds to the ENTH domainof HIP1.

[0019] The present invention further provides a composition comprising amutant HIP1 nucleic acid sequence lacking the ENTH domain. In someembodiments, the nucleic acid sequence comprises SEQ ID NO: 3. Thepresent invention also provides a composition comprising a polypeptideencoded by the nucleic acid sequence.

[0020] In some embodiments, the present invention provides the HIP1nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the presentinvention provides the polypeptide sequence of SEQ ID NO:2.

[0021] The present invention also provides a composition comprising amutant HIP1 polypeptide that induces cell death when expressed in acell. In some embodiments, the mutant HIP1 polypeptide is lacking a ENTHdomain. In some embodiments, the HIP1 polypeptide comprises SEQ ID NO:4. In some embodiments, the present invention provides a nucleic acidsequence encoding the polypeptide

[0022] The present invention additionally provides a non-humantransgenic animal lacking a functional HIP1 gene. In some embodiments,the non-human transgenic animal is a mouse. In some embodiments, theanimal comprises a knock-out of the HIP1 gene. In some embodiments, theknock-out is a conditional knock-out. In other embodiments, the animalcomprises a knock-in of the HIP1 gene.

[0023] In still further embodiments, the present invention provides acomposition comprising a drug, wherein the drug binds to wild type HIP1but not a HIP1 ENTH deletion mutant, and wherein the drug inhibits HIP1biological activity. In some embodiments, the drug binds to the ENTHdomain of HIP1. In some embodiments, the drug is a lipid analogue. Insome embodiments, the lipid analogue is a phosphoinositide mimetic.

DESCRIPTION OF THE FIGURES

[0024]FIG. 1 shows a Western blot analysis of the NCI 60 cancer-cellline screen.

[0025]FIG. 2 shows the results of immunohistochemical analysis of themulti-tumor tissue microarray TARPI.

[0026]FIG. 3 shows the results of immunohistochemical analysis of colonand prostate tumors.

[0027]FIG. 4 shows the relationship of HIP1 expression with clinicaloutcome in prostate cancer patients.

[0028]FIG. 5 shows a table of prostate tissue microarray analysisresults for 114 patients.

[0029]FIG. 6 shows HIP1 expression in a transgenic mouse model ofprostate cancer (TRAMP) (FIG. 6A) and effects of functional knockout ofHIP1 on cellular survival (FIG. 6B).

[0030]FIG. 7 shows the nucleic acid sequence of SEQ ID NO: 1.

[0031]FIG. 8 shows the amino acid sequence of SEQ ID NO: 2.

[0032]FIG. 9 shows the nucleic acid sequence of SEQ ID NO: 3.

[0033]FIG. 10 shows the amino acid sequence of SEQ ID NO: 4.

[0034]FIG. 11 shows the domain structure of HIP1.

[0035]FIG. 12 shows a graph of the rescue of apoptosis caused by HIP1ENTH deletion mutant with FLHIP1.

[0036]FIG. 13 shows a graph of the rescue of apoptosis caused by HIP1ENTH deletion mutant with Akt/Dncasp9.

[0037]FIG. 14 shows some HIP1 mutants of some embodiments of the presentinvention.

[0038]FIG. 15 shows the vector construction strategy for HIP1/PDGFβRknock-in.

[0039]FIG. 16 shows the vector construction strategy for HIP1conditional knock-out.

[0040]FIG. 17 shows the nucleic acid sequence of HIP1 untranslatedregions and Exons 1 and 2 (SEQ ID NO: 6).

[0041]FIG. 18 shows the vector construction strategy for the HIP1knock-out vector pHIP1KO.

[0042]FIG. 19 shows the deletion of the HIP1/PDGFβR knock-in ES cellallele.

GENERAL DESCRIPTION OF THE INVENTION

[0043] Originally identified as a protein that interacts with huntingtin(Kalchman et al., Nat.Genet. 16:44 [1997]; Wanker et al., Hum. Mol.Genet. 6:487 [1997]), HIP1 is a co-factor in clathrin mediatedtrafficking (Metzler et al., J. Biol. Chem. 276: 39271 [2001]; Mishra etal., JBC [2001]; Rao et al., Mol Cell Biol 21: 7796 [2001]; Waelter etal., Hum. Mol. Genet. 10: 1807 [2001]). The primary structure of HIP1predicts an epsin N-terminal homology (ENTH) domain, a leucine zippermotif and a carboxyl terminus homologous to TALIN. ENTH domains bind topolyphosphoinositide signaling lipids and have so far only been found inco-factors of clathrin mediated trafficking (Itoh et al., Science291:1047 [2001]; Ford et al., Science 291:1051 [2001]). Binding to lipidlocalizes the co-factors, including HIP1, to areas of receptor-mediatedendocytosis to assist in clathrin lattice formation.

[0044] The present invention is not limited to a particular mechanism.Indeed, an understanding of the mechanism is not necessary to practicethe present invention. Nonetheless, it is contemplated that HIP1expression promotes proliferation or cell survival by reducing thegrowth factor dependence of cells. HIP1 over-expression may dysregulategrowth factor receptor cell surface density or growth factor secretion,as a consequence of its role in clathrin-mediated trafficking.

[0045] The present invention demonstrates, using cancer cell lines,immunohistochemistry (IHC) and tissue microarrays, that HIP1 isspecifically over-expressed in colon and prostate tumors but is absentin normal prostatic and colonic epithelium. The expression of HIP1 inprostate cancer specimens from multiple patients was examined and foundto be expressed with an increased frequency in prostatic intraepithelialneoplasia (PIN), clinically localized prostate cancer (PCA), andmetastatic PCA. Expression of HIP1 in human prostate tumors wasevaluated in detail and correlated with progression of prostate cancer.This was evidenced by a lack of expression in the benign epitheliumcompared to the highest frequencies of expression in the most deadlycases of metastatic prostate cancer. In addition, there was a survivaladvantage in patients whose prostate confined prostate cancers did notexpress HIP1 compared to patients whose tumors did express HIP1. Thisindependent prognostication is exemplified by the patients with HIP1negative tumors who did well, in whom traditional pathologic indicators(stage and Gleason score) would have indicated a poor outcome. Its rolein tumorigenesis was further strengthened by its overexpression inprostate cancers in a mouse model of prostate cancer and data suggestingthat normal HIP1 expression maintains cell survival. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that HIP1 participates in theprogression of PCA.

[0046] In addition, it is contemplated that, similar to prostate cancer,the stratification of colonic adenocarcinomas based on HIP1 staining haspredictive value both with regard to tumor aggressiveness and patientsurvival. Because the vast majority of colonic tumors produce a similarhistologic picture it is difficult to obtain prognostic information fromhistologic analysis of tumor cells. Thus, the clinical outcome from alarge cohort of patients with colon cancer whose tumor HIP1 status isknown provides useful clinical information. Accordingly, the presentinvention provides a molecular marker whose expression is a valuableclinical tool for prognostication in the care of patients with two ofthe most common forms of cancer.

[0047] Definitions

[0048] To facilitate an understanding of the present invention, a numberof terms and phrases are defined below:

[0049] As used herein, the term “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (sIg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

[0050] As used herein, the term “antigen binding protein” refers toproteins that bind to a specific antigen. “Antigen binding proteins”include, but are not limited to, immunoglobulins, including polyclonal,monoclonal, chimeric, and humanized antibodies; Fab fragments, F(ab′)₂fragments, and Fab expression libraries; and single chain antibodies.

[0051] The term “epitope” as used herein refers to that portion of anantigen that makes contact with a particular immunoglobulin.

[0052] When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

[0053] The terms “specific binding” or “specifically binding” when usedin reference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0054] As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

[0055] As used herein, the term “a monoclonal antibody havingsubstantially the same properties as antibodies secreted by a hybridomaselected from the group consisting of those deposited as ATCC numberspending” refers to a monoclonal antibody having substantially the sameproperties as those disclosed in Example 1 (ATCC numbers pending),including but not limited to, specific binding to human and mouse HIP1protein and “specifically binds to HIP1 with low background binding.”

[0056] As used herein, the term “specifically binding to HIP1 with lowbackground binding” refers to an antibody that binds specifically toHIP1 protein (e.g., in an immunohistochemistry assay) but not to otherproteins (e.g., lack of non-specific binding).

[0057] As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

[0058] As used herein, the terms “epithelial tissue” or “epithelium”refer to the cellular covering of internal and external surfaces of thebody, including the lining of vessels and other small cavities. Itconsists of cells joined by small amounts of cementing substances.Epithelium is classified into types on the basis of the number of layersdeep and the shape of the superficial cells.

[0059] As used herein, the term “normal epithelium of prostate or colon”refers to prostate or colon epithelium that does not show any detectableindication of cancerous or pre-cancerous conditions.

[0060] As used herein, the term “cancerous epithelium of prostate orcolon” refers to prostate or colon epithelium that shows a detectableindication of cancerous or pre-cancerous conditions.

[0061] As used herein, the term “subject suspected of having cancer”refers to a subject that presents one or more symptoms indicative of acancer (e.g., a noticeable lump or mass) or is being screened for acancer (e.g., during a routine physical). A subject suspected of havingcancer may also have one or more risk factors. A subject suspected ofhaving cancer has generally not been tested for cancer. However, a“subject suspected of having cancer” encompasses an individual who hasreceived a preliminary diagnosis (e.g., a CT scan showing a mass orincreased PSA level) but for whom a confirmatory test (e.g., biopsyand/or histology) has not been done or for whom the stage of cancer isnot known. The term further includes people who once had cancer (e.g.,an individual in remission). A “subject suspected of having cancer” issometimes diagnosed with cancer and is sometimes found to not havecancer.

[0062] As used herein, the term “subject diagnosed with a cancer” refersto a subject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and antigen tests (e.g., PSA).

[0063] As used herein, the term “initial diagnosis” refers to a testresult of initial cancer diagnosis that reveals the presence or absenceof cancerous cells (e.g., using a biopsy and histology). An initialdiagnosis does not include information about the stage of the cancer orthe risk of prostate specific antigen failure.

[0064] As used herein, the term “prostate specific antigen failure”refers to the development of high prostate specific antigen levels in apatient following prostate cancer therapy (e.g., surgery).

[0065] As used herein, the term “risk of developing prostate specificantigen failure” refers to a subject's relative risk (e.g., the percentchance or a relative score) of developing prostate specific antigenfailure following prostate cancer therapy.

[0066] As used herein, the term “prostate tumor tissue” refers tocancerous tissue of the prostate. In some embodiments, the prostatetumor tissue is “post surgical prostate tumor tissue.”

[0067] As used herein, the term “post surgical tumor tissue” refers tocancerous tissue (e.g., prostate tissue) that has been removed from asubject (e.g., during surgery).

[0068] As used herein, the term “identifying the risk of said tumormetastasizing” refers to the relative risk (e.g., the percent chance ora relative score) of a tumor (e.g., prostate tumor tissue)metastasizing.

[0069] As used herein, the term “identifying the risk of said tumorrecurring” refers to the relative risk (e.g., the percent chance or arelative score) of a tumor (e.g., prostate tumor tissue) recurring inthe same organ as the original tumor (e.g., prostate).

[0070] As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental expose, and previous incidents of cancer,preexisting non-cancer diseases, and lifestyle.

[0071] As used herein, the term “characterizing cancer in subject”refers to the identification of one or more properties of a cancersample in a subject, including but not limited to, the presence ofbenign, pre-cancerous or cancerous tissue and the stage of the cancer.Cancers may be characterized by the identification of HIP1 in tumortissues.

[0072] As used herein, the term “characterizing tissue in a subject”refers to the identification of one or more properties of a tissuesample (e.g., including but not limited to, the presence of canceroustissue, the presence of pre-cancerous tissue that is likely to becomecancerous, and the presence of cancerous tissue that is likely tometastasize). In some embodiments, tissues are characterized by theidentification of the expression of HIP1.

[0073] As used herein, the term “reagent(s) capable of specificallydetecting HIP1 expression” refers to reagents used to detect theexpression of HIP1. Examples of suitable reagents include but are notlimited to, nucleic acid probes capable of specifically hybridizing toHIP1 mRNA or cDNA, and antibodies (e.g., monoclonal antibodies of thepresent invention).

[0074] As used herein, the term “instructions for using said kit fordetecting cancer in said subject” includes instructions for using thereagents contained in the kit for the detection and characterization ofcancer in a sample from a subject. In some embodiments, the instructionsfurther comprise the statement of intended use required by the U.S. Foodand Drug Administration (FDA) in labeling in vitro diagnostic products.The FDA classifies in vitro diagnostics as medical devices and requiredthat they be approved through the 510(k) procedure. Information requiredin an application under 510(k) includes: 1) The in vitro diagnosticproduct name, including the trade or proprietary name, the common orusual name, and the classification name of the device; 2) The intendeduse of the product; 3) The establishment registration number, ifapplicable, of the owner or operator submitting the 510(k) submission;the class in which the in vitro diagnostic product was placed undersection 513 of the FD&C Act, if known, its appropriate panel, or, if theowner or operator determines that the device has not been classifiedunder such section, a statement of that determination and the basis forthe determination that the in vitro diagnostic product is not soclassified; 4) Proposed labels, labeling and advertisements sufficientto describe the in vitro diagnostic product, its intended use, anddirections for use, including photographs or engineering drawings, whereapplicable; 5) A statement indicating that the device is similar toand/or different from other in vitro diagnostic products of comparabletype in commercial distribution in the U.S., accompanied by data tosupport the statement; 6) A 510(k) summary of the safety andeffectiveness data upon which the substantial equivalence determinationis based; or a statement that the 510(k) safety and effectivenessinformation supporting the FDA finding of substantial equivalence willbe made available to any person within 30 days of a written request; 7)A statement that the submitter believes, to the best of their knowledge,that all data and information submitted in the premarket notificationare truthful and accurate and that no material fact has been omitted;and 8) Any additional information regarding the in vitro diagnosticproduct requested that is necessary for the FDA to make a substantialequivalency determination. Additional information is available at theInternet web page of the U.S. FDA.

[0075] As used herein, the term “non-human transgenic animal lacking afunctional HIP1 gene” refers to a non-human animal (preferable a mammal,more preferably a mouse) whose endogenous HIP1 gene has been inactivated(e.g., as the result of a “HIP knockout” or a HIP1 knock-in”).

[0076] As used herein, the terms “HIP1 knockout” refers to a non-humananimal (e.g., a mouse) lacking a functional HIP1 gene. In someembodiments, the entire HIP1 gene is deleted. In other embodiments, thegene is inactivated via other means (e.g., deletion of essentialportions or inversions of some or all of the HIP1 gene). In otherembodiments, the HIP1 gene is inactivated using antisense inhibition.HIP1 knockout include conditional knockouts (e.g., selective inhibitionof gene activity). HIP1 knockout mice may be made using any suitablemethod including, but not limited to, those described herein. HIP1 genescan also be inactivated via the construction of a “HIP1 knock-in” inwhich the gene is inactivated by the insertion of exogenous DNA into aregion of the gene required for function.

[0077] As used herein, the term “ENTH domain of HIP1” refers to theepsin N-terminal homology domain of HIP1. In some embodiments, the ENTHdomain comprises amino acids 1-187 of SEQ ID NO: 2. The ENTH domain isbelieved to be responsible for HIP1 binding to polyphosphoinositollipids, which are involved in signaling. As used herein, the term “HIP1ENTH deletion mutant” refers to a HIP1 polypeptide that lacks theability to bind to polyphosphoinositol lipids (e.g., because of completeor partial deletion or mutation of the ENTH domain). In preferredembodiments, expression of “HIP1 ENTH deletion mutants” results in celldeath. One example of a “HIP1 ENTH deletion mutant” is the polypeptidedescribed by SEQ ID NO: 4.

[0078] As used herein, the term “lipid analogue” refers to a smallmolecule that has one or more structural features (e.g., a hydrophobicdomain) of a lipid (e.g., fatty acids). Lipid analogues includesmodified lipids, whether synthetic or naturally occurring.

[0079] As used herein, the term “mimetic” refers to a small moleculecompound that mimics the binding of a ligand (e.g., polyphosphoinositollipid) to HIP1. A mimetic that mimics the binding of apolyphosphoinositol lipid is referred to as a “polyphosphoinositolmimetic.” In some embodiments, mimetics are lipid analogues. Preferredmimetics bind to the ENTH domain of HIP1 and inhibit polyphosphoinositollipid signaling. Particularly preferred mimetics induce cell death incancer cells expressing HIP1 but not normal cells.

[0080] As used herein, the term “detecting a decrease in viability”refers to a decrease in the number of living cells in a culture. Inpreferred embodiments, the decrease is due to the induction ofprogrammed cell death (e.g., apoptosis) in some or all of the cells in apopulation.

[0081] As used herein, the term “induces cell death” refers to amolecule (e.g., a test compound or a drug) that induces a programmedcell death (e.g., apoptosis).

[0082] As used herein, the term “drug that inhibits HIP1 biologicalactivity” refers to a drug that inhibits one or more biologicalactivities of HIP1 (e.g., ligand binding and signaling). Preferred drugsare those that inhibit polyphosphoinositol lipid binding and subsequentsignaling.

[0083] As used herein, the terms “computer memory” and “computer memorydevice” refer to any storage media readable by a computer processor.Examples of computer memory include, but are not limited to, RAM, ROM,computer chips, digital video disc (DVDs), compact discs (CDs), harddisk drives (HDD), and magnetic tape.

[0084] As used herein, the term “computer readable medium” refers to anydevice or system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

[0085] As used herein, the terms “processor” and “central processingunit” or “CPU” are used interchangeably and refer to a device that isable to read a program from a computer memory (e.g., ROM or othercomputer memory) and perform a set of steps according to the program.

[0086] As used herein, the term “stage of cancer” refers to aqualitative or quantitative assessment of the level of advancement of acancer. Criteria used to determine the stage of a cancer include, butare not limited to, the size of the tumor, whether the tumor has spreadto other parts of the body and where the cancer has spread (e.g., withinthe same organ or region of the body or to another organ).

[0087] As used herein, the term “providing a prognosis” refers toproviding information regarding the impact of the presence of cancer(e.g., as determined by the diagnostic methods of the present invention)on a subject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

[0088] As used herein, the term “non-human animals” refers to allnon-human animals including, but are not limited to, vertebrates such asrodents, non-human primates, ovines, bovines, ruminants, lagomorphs,porcines, caprines, equines, canines, felines, aves, etc.

[0089] As used herein, the term “gene transfer system” refers to anymeans of delivering a composition comprising a nucleic acid sequence toa cell or tissue. For example, gene transfer systems include, but arenot limited to, vectors (e.g., retroviral, adenoviral, adeno-associatedviral, and other nucleic acid-based delivery systems), microinjection ofnaked nucleic acid, polymer-based delivery systems (e.g., liposome-basedand metallic particle-based systems), biolistic injection, and the like.As used herein, the term “viral gene transfer system” refers to genetransfer systems comprising viral elements (e.g., intact viruses,modified viruses and viral components such as nucleic acids or proteins)to facilitate delivery of the sample to a desired cell or tissue. Asused herein, the term “adenovirus gene transfer system” refers to genetransfer systems comprising intact or altered viruses belonging to thefamily Adenoviridae.

[0090] As used herein, the term “site-specific recombination targetsequences” refers to nucleic acid sequences that provide recognitionsequences for recombination factors and the location where recombinationtakes place.

[0091] As used herein, the term “nucleic acid molecule” refers to anynucleic acid containing molecule, including but not limited to, DNA orRNA. The term encompasses sequences that include any of the known baseanalogs of DNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0092] The term “gene” refers to a nucleic acid (e.g., DNA) sequencethat comprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

[0093] As used herein, the term “heterologous gene” refers to a genethat is not in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

[0094] As used herein, the term “transgene” refers to a heterologousgene that is integrated into the genome of an organism (e.g., anon-human animal) and that is transmitted to progeny of the organismduring sexual reproduction.

[0095] As used herein, the term “transgenic organism” refers to anorganism (e.g., a non-human animal) that has a transgene integrated intoits genome and that transmits the transgene to its progeny during sexualreproduction.

[0096] As used herein, the term “gene expression” refers to the processof converting genetic information encoded in a gene into RNA (e.g.,mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e.,via the enzymatic action of an RNA polymerase), and for protein encodinggenes, into protein through “translation” of mRNA. Gene expression canbe regulated at many stages in the process. “Up-regulation” or“activation” refers to regulation that increases the production of geneexpression products (i.e., RNA or protein), while “down-regulation” or“repression” refers to regulation that decrease production. Molecules(e.g., transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

[0097] In addition to containing introns, genomic forms of a gene mayalso include sequences located on both the 5′ and 3′ end of thesequences that are present on the RNA transcript. These sequences arereferred to as “flanking” sequences or regions (these flanking sequencesare located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

[0098] The term “wild-type” refers to a gene or gene product isolatedfrom a naturally occurring source. A wild-type gene is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene. In contrast, theterm “modified” or “mutant” refers to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally occurring mutants can be isolated;these are identified by the fact that they have altered characteristics(including altered nucleic acid sequences) when compared to thewild-type gene or gene product.

[0099] As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

[0100] As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotideor polynucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

[0101] As used herein, the term “oligonucleotide,” refers to a shortlength of single-stranded polynucleotide chain. Oligonucleotides aretypically less than 200 residues long (e.g., between 15 and 100),however, as used herein, the term is also intended to encompass longerpolynucleotide chains. Oligonucleotides are often referred to by theirlength. For example a 24 residue oligonucleotide is referred to as a“24-mer”. Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

[0102] As used herein, the terms “complementary” or “complementarity”are used in reference to polynucleotides (i.e., a sequence ofnucleotides) related by the base-pairing rules. For example, for thesequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

[0103] The term “homology” refers to a degree of complementarity. Theremay be partial homology or complete homology (i.e., identity). Apartially complementary sequence is a nucleic acid molecule that atleast partially inhibits a completely complementary nucleic acidmolecule from hybridizing to a target nucleic acid is “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous nucleic acidmolecule to a target under conditions of low stringency. This is not tosay that conditions of low stringency are such that non-specific bindingis permitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget that is substantially non-complementary (e.g., less than about30% identity); in the absence of non-specific binding the probe will nothybridize to the second non-complementary target.

[0104] When used in reference to a double-stranded nucleic acid sequencesuch as a cDNA or genomic clone, the term “substantially homologous”refers to any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

[0105] A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

[0106] When used in reference to a single-stranded nucleic acidsequence, the term “substantially homologous” refers to any probe thatcan hybridize (i.e., it is the complement of) the single-strandednucleic acid sequence under conditions of low stringency as describedabove.

[0107] As used herein, the term “hybridization” is used in reference tothe pairing of complementary nucleic acids. Hybridization and thestrength of hybridization (i.e., the strength of the association betweenthe nucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

[0108] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theT_(m) of nucleic acids is well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (See e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization[1985]). Other references include more sophisticated computations thattake structural as well as sequence characteristics into account for thecalculation of T_(m).

[0109] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under ‘medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely relation sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

[0110] “High stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

[0111] “Medium stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

[0112] “Low stringency conditions” comprise conditions equivalent tobinding or hybridization at 42° C. in a solution consisting of 5×SSPE(43.8 g/l NaCl, 6.9 g/l NaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's containsper 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V;Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing ina solution comprising 5×SSPE, 0.1 % SDS at 42° C. when a probe of about500 nucleotides in length is employed.

[0113] The art knows well that numerous equivalent conditions may beemployed to comprise low stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (e.g., increasing the temperature of thehybridization and/or wash steps, the use of formamide in thehybridization solution, etc.) (see definition above for “stringency”).

[0114] “Amplification” is a special case of nucleic acid replicationinvolving template specificity. It is to be contrasted with non-specifictemplate replication (i.e., replication that is template-dependent butnot dependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

[0115] Template specificity is achieved in most amplification techniquesby the choice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]).Other nucleic acids will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlin et al.,Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu and Wallace, Genomics 4:560[1989]). Finally, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press[1989]).

[0116] As used herein, the term “amplifiable nucleic acid” is used inreference to nucleic acids that may be amplified by any amplificationmethod. It is contemplated that “amplifiable nucleic acid” will usuallycomprise “sample template.”

[0117] As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target.”In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

[0118] As used herein, the term “primer” refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, that is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product that is complementary to a nucleic acidstrand is induced, (i.e., in the presence of nucleotides and an inducingagent such as DNA polymerase and at a suitable temperature and pH). Theprimer is preferably single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

[0119] As used herein, the term “probe” refers to an oligonucleotide(i.e., a sequence of nucleotides), whether occurring naturally as in apurified restriction digest or produced synthetically, recombinantly orby PCR amplification, that is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

[0120] As used herein, the term “target,” refers to the region ofnucleic acid bounded by the primers. Thus, the “target” is sought to besorted out from other nucleic acid sequences. A “segment” is defined asa region of nucleic acid within the target sequence.

[0121] As used herein, the term “polymerase chain reaction” (“PCR”)refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 4,683,202,and 4,965,188, hereby incorporated by reference, which describe a methodfor increasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

[0122] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process are,themselves, efficient templates for subsequent PCR amplifications.

[0123] As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

[0124] As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

[0125] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0126] The terms “in operable combination,” “in operable order,” and“operably linked” as used herein refer to the linkage of nucleic acidsequences in such a manner that a nucleic acid molecule capable ofdirecting the transcription of a given gene and/or the synthesis of adesired protein molecule is produced. The term also refers to thelinkage of amino acid sequences in such a manner so that a functionalprotein is produced.

[0127] The term “isolated” when used in relation to a nucleic acid, asin “an isolated oligonucleotide” or “isolated polynucleotide” refers toa nucleic acid sequence that is identified and separated from at leastone component or contaminant with which it is ordinarily associated inits natural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

[0128] As used herein, the term “purified” or “to purify” refers to theremoval of components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

[0129] “Amino acid sequence” and terms such as “polypeptide” or“protein” are not meant to limit the amino acid sequence to thecomplete, native amino acid sequence associated with the recited proteinmolecule.

[0130] The term “native protein” as used herein to indicate that aprotein does not contain amino acid residues encoded by vectorsequences; that is, the native protein contains only those amino acidsfound in the protein as it occurs in nature. A native protein may beproduced by recombinant means or may be isolated from a naturallyoccurring source.

[0131] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid.

[0132]

[0133] The term “Southern blot,” refers to the analysis of DNA onagarose or acrylamide gels to fractionate the DNA according to sizefollowed by transfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, N.Y., pp 9.31-9.58 [1989]).

[0134] The term “Northern blot,” as used herein refers to the analysisof RNA by electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

[0135] The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

[0136] As used herein, the term “vector” is used in reference to nucleicacid molecules that transfer DNA segment(s) from one cell to another.The term “vehicle” is sometimes used interchangeably with “vector.”Vectors are often derived from plasmids, bacteriophages, or plant oranimal viruses.

[0137] The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

[0138] The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher (or greater) than thatobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA are measured using any of a number of techniques known to thoseskilled in the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the mRNA-specificsignal observed on Northern blots). The amount of mRNA present in theband corresponding in size to the correctly spliced transgene RNA isquantified; other minor species of RNA which hybridize to the transgeneprobe are not considered in the quantification of the expression of thetransgenic mRNA.

[0139] The term “transfection” as used herein refers to the introductionof foreign DNA into eukaryotic cells. Transfection may be accomplishedby a variety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

[0140] The term “calcium phosphate co-precipitation” refers to atechnique for the introduction of nucleic acids into a cell. The uptakeof nucleic acids by cells is enhanced when the nucleic acid is presentedas a calcium phosphate-nucleic acid co-precipitate. The originaltechnique of Graham and van der Eb (Graham and van der Eb, Virol.,52:456 [1973]), has been modified by several groups to optimizeconditions for particular types of cells. The art is well aware of thesenumerous modifications.

[0141] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

[0142] The term “transient transfection” or “transiently transfected”refers to the introduction of foreign DNA into a cell where the foreignDNA fails to integrate into the genome of the transfected cell. Theforeign DNA persists in the nucleus of the transfected cell for severaldays. During this time the foreign DNA is subject to the regulatorycontrols that govern the expression of endogenous genes in thechromosomes. The term “transient transfectant” refers to cells that havetaken up foreign DNA but have failed to integrate this DNA.

[0143] As used herein, the term “selectable marker” refers to the use ofa gene that encodes an enzymatic activity that confers the ability togrow in medium lacking what would otherwise be an essential nutrient(e.g. the HIS3 gene in yeast cells); in addition, a selectable markermay confer resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed. Selectable markers may be“dominant”; a dominant selectable marker encodes an enzymatic activitythat can be detected in any eukaryotic cell line. Examples of dominantselectable markers include the bacterial aminoglycoside 3′phosphotransferase gene (also referred to as the neo gene) that confersresistance to the drug G418 in mammalian cells, the bacterial hygromycinG phosphotransferase (hyg) gene that confers resistance to theantibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) that confers theability to grow in the presence of mycophenolic acid. Other selectablemarkers are not dominant in that their use must be in conjunction with acell line that lacks the relevant enzyme activity. Examples ofnon-dominant selectable markers include the thymidine kinase (tk) genethat is used in conjunction with tk⁻ cell lines, the CAD gene that isused in conjunction with CAD-deficient cells and the mammalianhypoxanthine-guanine phosphoribosyl transferase (hprt) gene that is usedin conjunction with hprt⁻ cell lines. A review of the use of selectablemarkers in mammalian cell lines is provided in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, N.Y. (1989) pp.16.9-16.15.

[0144] As used herein, the term “cell culture” refers to any in vitroculture of cells. Included within this term are continuous cell lines(e.g., with an immortal phenotype), primary cell cultures, transformedcell lines, finite cell lines (e.g., non-transformed cells), and anyother cell population maintained in vitro.

[0145] As used, the term “eukaryote” refers to organisms distinguishablefrom “prokaryotes.” It is intended that the term encompass all organismswith cells that exhibit the usual characteristics of eukaryotes, such asthe presence of a true nucleus bounded by a nuclear membrane, withinwhich lie the chromosomes, the presence of membrane-bound organelles,and other characteristics commonly observed in eukaryotic organisms.Thus, the term includes, but is not limited to such organisms as fungi,protozoa, and animals (e.g., humans).

[0146] As used herein, the term “in vitro” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment. In vitro environments can consist of, but arenot limited to, test tubes and cell culture. The term “in vivo” refersto the natural environment (e.g., an animal or a cell) and to processesor reaction that occur within a natural environment.

[0147] The terms “test compound” and “candidate compound” refer to anychemical entity, pharmaceutical, drug, and the like that is a candidatefor use to treat or prevent a disease, illness, sickness, or disorder ofbodily function (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention.

[0148] As used herein, the term “sample” is used in its broadest sense.In one sense, it is meant to include a specimen or culture obtained fromany source, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0149] The present invention provides novel antibodies to HIP1. Thepresent invention further provides methods for providing cancer (e.g.,colon and prostate cancers) diagnoses, prognoses, and treatments (e.g.,using the monoclonal antibodies of the present invention). The presentinvention further provides methods of screening potential therapeuticcompounds for HIP1 inhibitory properties.

[0150] I. HIP1 as a Marker for Cancer

[0151] The present invention relates to compositions and methods forcancer diagnostics, including but not limited to, HIP1 cancer markers.In particular, the present invention provides markers (e.g., HIP1) whoseexpression is specifically altered in cancerous tissues (e.g.,epithelial tissues such as prostate and colon). Such markers find use inthe diagnosis and characterization of cancer.

[0152] A. Identification of Markers

[0153] Experiments conducted during the development of the presentinvention resulted in the generation of highly specific and sensitivemonoclonal antibodies against Huntingtin Interacting Protein 1 (HIP1).Three of the anti-HIP1 antibodies were used to investigate the presenceof HIP1 in cancer tissues. HIP1 was found to be strongly expressed in amajority of prostate cancer and colon cancer specimens, but rarely andweakly expressed in normal prostate and colon tissue. Accordingly, thepresent invention provides methods for diagnosing cancers (e.g.,prostate and colon cancers) by detecting the presence of HIP1. Inaddition, increased HIP1 expression was found to correlate with anegative prognosis.

[0154] B. HIP1 Mutants

[0155] The present invention further provides HIP1 mutants. Most HIP1mutants can be expressed in combination with endogenous HIP1 withoutseriously affecting cell survival or proliferation. However, a constructidentified during the development of the present invention caused cellsto undergo cell death upon expression. The mutant lacked the ENTH domainof HIP1 (See FIG. 11 for a description of the domain structure of HIP1).The HIP1 ENTH deletion mutant generated during the development of thepresent invention is described in Example 3 and SEQ ID NOs: 3 and 4. TheENTH domain is responsible for HIP1 binding to polyphosphoinositollipids, which act as signaling molecules. The present invention is notlimited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that the ENTH deletion mutants disruptHIP1's normal role of linking clathrin mediated trafficking tosignaling. In addition, over-expression of wild-type HIP1 prevented theinduction of cell death by the ENTH deletion mutant. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention.

[0156] The present invention is not limited to the ENTH deletion mutantof SEQ ID NOs: 3 and 4. The present invention contemplates any mutationthat alters or inhibits the normal biological function of HIP1. In someembodiments, the HIP1 mutant is one of the mutants described in FIG. 14.In some preferred embodiments, the mutation results in the inactivationof the ENTH domain (e.g., as determined by the ability of the mutant toinduce cell death when expressed). Preferred ENTH deletion mutants areinduce cell death when expressed alone but the induction of cell deathis prevented by over-expression of a wild-type HIP1. HIP1 mutants can begenerated using any suitable method including, but not limited to,standard molecular biology techniques well known in the art or byisolation of naturally occurring mutants.

[0157] C. Detection of HIP1

[0158] In some embodiments, the present invention provides methods fordetection of HIP1. In preferred embodiments, the presence of HIP1protein or mRNA is measured directly. In some embodiments, HIP1 mRNA orprotein is detected in tissue samples (e.g., biopsy samples). In otherembodiments, HIP1 mRNA or protein is detected in bodily fluids (e.g.,serum, plasma, or urine). The present invention further provides kitsfor the detection of HIP1. In preferred embodiments, the presence ofHIP1 is used to provide a diagnosis or prognosis to a subject.

[0159] In some preferred embodiments, HIP1 protein is detected. Proteinexpression may be detected by any suitable method. In some embodiments,proteins are detected by binding of an antibody specific for theprotein. In some preferred embodiments, the monoclonal antibodydescribed in Example 1 below is utilized. The present invention is notlimited to a particular antibody. Any antibody (monoclonal orpolyclonal) that specifically detects HIP1 may by utilized. Methods forthe generation of antibodies are described below.

[0160] Antibody binding is detected by techniques known in the art. Forexample, in some embodiments where HIP1 protein is detected in bodilyfluids, antibody binding is detected using a suitable technique,including but not limited to, radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotope labels,for example), Western blots, precipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays, etc.),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays. In other embodiments, where HIP1protein is detected in tissue samples, immunohistochemistry is utilizedfor the detection of antibody binding.

[0161] In one embodiment, antibody binding is detected by detecting alabel on the primary antibody. In another embodiment, the primaryantibody is detected by detecting binding of a secondary antibody orreagent to the primary antibody. In a further embodiment, the secondaryantibody is labeled. Many methods are known in the art for detectingbinding in an immunoassay and are within the scope of the presentinvention.

[0162] In some embodiments, an automated detection assay is utilized.Methods for the automation of immunoassays include, but are not limitedto, those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750,and 5,358,691, each of which is herein incorporated by reference. Insome embodiments, the analysis and presentation of results is alsoautomated. For example, in some embodiments, software that generates adiagnosis and/or prognosis based on the presence or absence of a seriesof proteins corresponding to cancer markers is utilized.

[0163] In other embodiments, the immunoassay described in U.S. Pat. Nos.5,599,677 and 5,672,480, each of which is herein incorporated byreference, is utilized. In other embodiments, proteins are detected byimmunohistochemistry.

[0164] In other embodiments, HIP1 is detected at the level of HIP1 RNA.In some embodiments, HIP1 RNA is detected by measuring the expression ofcorresponding mRNA in a tissue sample (e.g., prostate or colon tissue).mRNA expression may be measured by any suitable method, including butnot limited to, those disclosed below.

[0165] In some embodiments, RNA is detected by Northern blot analysis.Northern blot analysis involves the separation of RNA and hybridizationof a complementary labeled probe. Methods for Northern blot analysis arewell known in the art.

[0166] In other embodiments, RNA expression is detected by enzymaticcleavage of specific structures (INVADER assay, Third Wave Technologies;See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and5,994,069; each of which is herein incorporated by reference). TheINVADER assay detects specific nucleic acid (e.g., RNA) sequences byusing structure-specific enzymes to cleave a complex formed by thehybridization of overlapping oligonucleotide probes.

[0167] In still further embodiments, RNA (or corresponding cDNA) isdetected by hybridization to a oligonucleotide probe. A variety ofhybridization assays using a variety of technologies for hybridizationand detection are available. For example, in some embodiments, TaqManassay (Applied Biosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos.5,962,233 and 5,538,848, each of which is herein incorporated byreference) is utilized. The assay is performed during a PCR reaction.The TaqMan assay exploits the 5′-3′ exonuclease activity of the AMPLITAQGOLD DNA polymerase. A probe consisting of an oligonucleotide with a5′-reporter dye (e.g., a fluorescent dye) and a 3′-quencher dye isincluded in the PCR reaction. During PCR, if the probe is bound to its,target, the 5′-3′ nucleolytic activity of the AMPLITAQ GOLD polymerasecleaves the probe between the reporter and the quencher dye. Theseparation of the reporter dye from the quencher dye results in anincrease of fluorescence. The signal accumulates with each cycle of PCRand can be monitored with a fluorimeter.

[0168] In yet other embodiments, reverse-transcriptase PCR (RT-PCR) isused to detect the expression of RNA. In RT-PCR, RNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a template for a PCR reaction. PCRproducts can be detected by any suitable method, including but notlimited to, gel electrophoresis and staining with a DNA specific stainor hybridization to a labeled probe. In some embodiments, thequantitative reverse transcriptase PCR with standardized mixtures ofcompetitive templates method described in U.S. Pat. Nos. 5,639,606,5,643,765, and 5,876,978 (each of which is herein incorporated byreference) is utilized.

[0169] D. Kits

[0170] In some embodiments, the present invention provides kits for thedetection and characterization of cancer (e.g., prostate and coloncancer). In some embodiments, the kits contain antibodies specific forHIP1, in addition to detection reagents and buffers. In otherembodiments, the kits contain reagents specific for the detection ofHIP1 mRNA or cDNA (e.g., oligonucleotide probes or primers). Inpreferred embodiments, the kits contain all of the components necessaryto perform a detection assay, including all controls, directions forperforming assays, and any necessary software for analysis andpresentation of results.

[0171] II. Antibodies

[0172] The present invention provides isolated antibodies. In preferredembodiments, the present invention provides monoclonal antibodies thatspecifically bind to an isolated polypeptide comprised of at least fiveamino acid residues of HIP1. These antibodies find use in the diagnosticmethods described herein.

[0173] An antibody against a protein of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

[0174] The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

[0175] For preparing monoclonal antibody-producing cells, an individualanimal whose antibody titer has been confirmed (e.g., a mouse) isselected, and 2 days to 5 days after the final immunization, its spleenor lymph node is harvested and antibody-producing cells containedtherein are fused with myeloma cells to prepare the desired monoclonalantibody producer hybridoma. Measurement of the antibody titer inantiserum can be carried out, for example, by reacting the labeledprotein, as described hereinafter and antiserum and then measuring theactivity of the labeling agent bound to the antibody. The cell fusioncan be carried out according to known methods, for example, the methoddescribed by Koehler and Milstein (Nature 256:495 [1975]). As a fusionpromoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ),preferably PEG is used.

[0176] Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and thelike. The proportion of the number of antibody producer cells (spleencells) and the number of myeloma cells to be used is preferably about1:1 to about 20:1. PEG (preferably PEG 1000-PEG 6000) is preferablyadded in concentration of about 10% to about 80%. Cell fusion can becarried out efficiently by incubating a mixture of both cells at about20° C. to about 40° C., preferably about 30° C. to about 37° C. forabout 1 minute to 10 minutes.

[0177] Various methods may be used for screening for a hybridomaproducing the antibody (e.g., against HIP1). For example, where asupernatant of the hybridoma is added to a solid phase (e.g.,microplate) to which antibody is adsorbed directly or together with acarrier and then an anti-immunoglobulin antibody (if mouse cells areused in cell fusion, anti-mouse immunoglobulin antibody is used) orProtein A labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase. Alternately, a supernatant of the hybridoma is added to a solidphase to which an anti-immunoglobulin antibody or Protein A is adsorbedand then the protein labeled with a radioactive substance or an enzymeis added to detect the monoclonal antibody against the protein bound tothe solid phase.

[0178] Selection of the monoclonal antibody can be carried out accordingto any known method or its modification. Normally, a medium for animalcells to which HAT (hypoxanthine, aminopterin, thymidine) are added isemployed. Any selection and growth medium can be employed as long as thehybridoma can grow. For example, RPMI 1640 medium containing 1% to 20%,preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to10% fetal bovine serum, a serum free medium for cultivation of ahybridoma (SFM-101, Nissui Seiyaku) and the like can be used. Normally,the cultivation is carried out at 20° C. to 40° C., preferably 37° C.for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5%CO₂ gas. The antibody titer of the supernatant of a hybridoma culturecan be measured according to the same manner as described above withrespect to the antibody titer of the anti-protein in the antiserum.

[0179] Separation and purification of a monoclonal antibody (e.g.,against HIP1) can be carried out according to the same manner as thoseof conventional polyclonal antibodies such as separation andpurification of immunoglobulins, for example, salting-out, alcoholicprecipitation, isoelectric point precipitation, electrophoresis,adsorption and desorption with ion exchangers (e.g., DEAE),ultracentrifugation, gel filtration, or a specific purification methodwherein only an antibody is collected with an active adsorbent such asan antigen-binding solid phase, Protein A or Protein G and dissociatingthe binding to obtain the antibody.

[0180] Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody against is recovered from the immunized animal and the antibodyis separated and purified.

[0181] As to the complex of the immunogen and the carrier protein to beused for immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

[0182] In addition, various condensing agents can be used for couplingof a hapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

[0183] The polyclonal antibody is recovered from blood, ascites and thelike, of an animal immunized by the above method. The antibody titer inthe antiserum can be measured according to the same manner as thatdescribed above with respect to the supernatant of the hybridomaculture. Separation and purification of the antibody can be carried outaccording to the same separation and purification method ofimmunoglobulin as that described with respect to the above monoclonalantibody.

[0184] The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, HIP1 protein (furtherincluding a gene having a nucleotide sequence partly altered) can beused as the immunogen. Further, fragments of the protein may be used.Fragments may be obtained by any methods including, but not limited toexpressing a fragment of the gene, enzymatic processing of the protein,chemical synthesis, and the like.

[0185] In some embodiments, antibodies (e.g., monoclonal antibodies) arehumanized. Such humanized antibodies find particular use in the cancerimmunotherapies described below. Humanized antibodies are altered inorder to make them less immunogenic to humans, e.g., by constructingchimeric antibodies in which a mouse antigen-binding variable domain iscoupled to a human constant domain. Humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies. Methods for humanizing antibodies are well known in the artand include but are not limited to, those disclosed in U.S. Pat. Nos.6,054,297, 4,816,567, 6,180,377, 5,871,907, 5,585,089, and 6,180,370,each of which is herein incorporated by reference.

[0186] III. Drug Screening

[0187] In some embodiments, the present invention provides drugscreening assays (e.g., to screen for anticancer drugs). The screeningmethods of the present invention utilize HIP1. For example, in someembodiments, the present invention provides methods of screening forcompounds that alter (e.g., increase or decrease) the expression ofHIP1. In some embodiments, candidate compounds are antisense agents(e.g., oligonucleotides) directed against HIP1. See Section IV below fora discussion of antisense therapy. In other embodiments, candidatecompounds are antibodies (e.g., those described in Section II above andthe below Example. In other embodiments, candidate compounds are smallmolecules. In some embodiments, given the fact that the ENTH deletionHIP1 construct has the phosphoinositide binding site of HIP1 deleted,lipid analogues are utilized as candidate compounds. In otherembodiments, non-lipid analogue small molecules are utilized ascandidate compounds.

[0188] A. HIP1 Expression Assays

[0189] In one screening method, candidate compounds are evaluated fortheir ability to alter HIP1 expression by contacting a compound with acell expressing HIP1 and then assaying for the effect of the candidatecompounds on expression. In some embodiments, the effect of candidatecompounds on expression of HIP1 is assayed for by detecting the level ofcancer marker mRNA expressed by the cell. mRNA expression can bedetected by any suitable method, including but not limited to, thosedisclosed herein.

[0190] In other embodiments, the effect of candidate compounds isassayed by measuring the level of HIP1 expression. The level ofpolypeptide expressed can be measured using any suitable method,including but not limited to, those disclosed herein.

[0191] B. Cellular Assays

[0192] In some preferred embodiments, the present invention providesmethods of identifying small molecule inhibitors of HIP1. In someembodiments, methods are utilized that identify compounds that mimic theeffect of the ENTH deletion mutant described in Example 3 (e.g., causecell death). For example, in some embodiments, a high throughputscreening method comprising the use of HIP1+embryonic fibroblastsobtained from wild-type mice and HIP1−/−embryonic fibroblasts obtainedfrom HIP1 knockout mice is utilized (See e.g., Example 5). Thesefibroblasts are cultured and libraries of therapeutic compounds areadded to the cultures. Specific inhibitors of HIP1 are then identifiedas compounds that kill the HIP1+fibroblasts but not theHIP1−fibroblasts.

[0193] In other embodiments, a screen against human HIP1 is performed byusing HIP1 expressing cancer cell lines (e.g., the majority of cancercell lines) and HIP1 non-expressing human cancer cell lines (e.g.,colo205 cell lines, ATCC; (See e.g., Example 5)). Such cell lines arecultured in the presence of a library of therapeutic compounds toidentify compounds that kill HIP1+ but not HIP1−cell lines. Thesemethods represent effective ways of screening for therapeutic compoundsagainst a variety of epithelial cancers.

[0194] C. In Vitro Assays

[0195] In some embodiments, In vitro drug screens are performed usingpurified wild type HIP1 and the HIP1 ENTH deletion mutant described inExample 3. In some embodiments, the HIP1 proteins are immobilized tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.Binding of a test compound to HIP1 or HIP1 ENTH deletion mutant can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicrocentrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/AIP-6fusion proteins or glutathione-S-transferase/target fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and thenon-adsorbed protein, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of protein binding or activity determined using standardtechniques.

[0196] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, HIP1 orHIP1 ENTH deletion mutant can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated HIP1 or HIP1 ENTH deletion can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with HIP1or HIP1 ENTH deletion mutants but which do not interfere with binding ofthe protein to test compounds can be derivatized to the wells of theplate, and unbound protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with HIP1 or HIP1 ENTH deletionmutants, as well as enzyme-linked assays that rely on detecting anenzymatic activity associated with the HIP1 or HIP1 ENTH deletionmutant.

[0197] In other embodiments, a competitive drug screening assays inwhich neutralizing antibodies capable of binding wt HIP1 specificallycompete with a test compound for binding HIP1 are utilized. In thismanner, the antibodies can be used to detect the presence of anycompound that shares one or more antigenic determinants with HIP1.

[0198] D. In vivo Assays

[0199] In still further embodiments, transgenic animals having altered(e.g., inactivated or overexpressed) HIP1 gene are utilized in drugscreening applications. Exemplary transgenic animals are described belowand in Example 4. For example, in some embodiments, compounds arescreened for their ability to reduce tumors in wild type mice but notHIP1 deletion mutants. In some preferred embodiments, HIP1 conditionalknock-out mice are utilized in such an assay. In some embodiments, theconditional HIP1 mutants described in Example 4 are utilized. These micehave a functional HIP1 gene that can be inactivated via theadministration of an adenoviral vector containing the cre gene.

[0200] In other embodiments, transgenic animals that overexpress HIP1 intissues that typically have HIP1+tumors (e.g., breast, prostate, andcolon) are utilized for drug screening. Such mice are administeredlibraries of compounds and a decrease in tumor size or lack ofexpression of HIP1 is screened for.

[0201] In still further embodiments, the mouse model of prostate cancerdescribed in Example 2 is utilized in drug screening applications. Suchmice are administered libraries of compounds and a decrease in tumorsize is screened for.

[0202] IV. Cancer Therapies

[0203] In some embodiments, the present invention provides therapies forcancer (e.g., prostate and colon cancer). In some embodiments, therapiestarget HIP1.

[0204] A. Antisense Therapies

[0205] In some embodiments, the present invention targets the expressionof HIP1. For example, in some embodiments, the present invention employscompositions comprising oligomeric antisense compounds, particularlyoligonucleotides (e.g., those identified in the drug screening methodsdescribed above), for use in modulating the function of nucleic acidmolecules encoding HIP1, ultimately modulating the amount of HIP1expressed. This is accomplished by providing antisense compounds thatspecifically hybridize with one or more nucleic acids encoding HIP1. Thespecific hybridization of an oligomeric compound with its target nucleicacid interferes with the normal function of the nucleic acid. Thismodulation of function of a target nucleic acid by compounds thatspecifically hybridize to it is generally referred to as “antisense.”The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include allvital functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity that may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the expression of HIP1. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. For example,expression may be inhibited to potentially prevent tumor proliferation.

[0206] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of the present invention, is a multistep process. The processusually begins with the identification of a nucleic acid sequence whosefunction is to be modulated. This may be, for example, a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In the present invention, the target is a nucleicacid molecule encoding HIP1. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon.” A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the present invention, “startcodon” and “translation initiation codon” refer to the codon or codonsthat are used in vivo to initiate translation of an mRNA moleculetranscribed from a gene encoding a tumor antigen of the presentinvention, regardless of the sequence(s) of such codons.

[0207] Translation termination codon (or “stop codon”) of a gene mayhave one of three sequences (i.e., 5′-UAA, 5′-UAG and 5′-UGA; thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively). The terms “start codon region” and “translationinitiation codon region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationcodon region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation termination codon.

[0208] The open reading frame (ORF) or “coding region,” which refers tothe region between the translation initiation codon and the translationtermination codon, is also a region that may be targeted effectively.Other target regions include the 5′ untranslated region (5′ UTR),referring to the portion of an mRNA in the 5′ direction from thetranslation initiation codon, and thus including nucleotides between the5′ cap site and the translation initiation codon of an mRNA orcorresponding nucleotides on the gene, and the 3′ untranslated region(3′ UTR), referring to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. The 5′ cap of an mRNA comprisesan N7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The cap region may also be apreferred target region.

[0209] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” thatare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites (i.e.,intron-exon junctions) may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0210] Once one or more target sites have been identified,oligonucleotides are chosen that are sufficiently complementary to thetarget (i.e., hybridize sufficiently well and with sufficientspecificity) to give the desired effect. For example, in preferredembodiments of the present invention, antisense oligonucleotides aretargeted to or near the start codon.

[0211] In the context of this invention, “hybridization,” with respectto antisense compositions and methods, means hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases. For example,adenine and thymine are complementary nucleobases that pair through theformation of hydrogen bonds. It is understood that the sequence of anantisense compound need not be 100% complementary to that of its targetnucleic acid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired (i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed).

[0212] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with specificity, can be used to elucidate thefunction of particular genes. Antisense compounds are also used, forexample, to distinguish between functions of various members of abiological pathway.

[0213] The specificity and sensitivity of antisense is also applied fortherapeutic uses. For example, antisense oligonucleotides have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man. Antisense oligonucleotides have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides areuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues, and animals,especially humans.

[0214] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about30 nucleobases (i.e., from about 8 to about 30 linked bases), althoughboth longer and shorter sequences may find use with the presentinvention. Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 25 nucleobases.

[0215] Specific examples of preferred antisense compounds useful withthe present invention include oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. As defined in thisspecification, oligonucleotides having modified backbones include thosethat retain a phosphorus atom in the backbone and those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, modified oligonucleotides that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

[0216] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0217] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0218] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage (i.e., the backbone) of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science 254:1497 (1991).

[0219] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂, —NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0220] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta 78:486 [1995]) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy (i.e., a O(CH₂)₂ON(CH₃)₂group), also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (alsoknown in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

[0221] Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

[0222] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2.degree ° C. and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

[0223] Another modification of the oligonucleotides of the presentinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates that enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, analiphatic chain, (e.g., dodecandiol or undecyl residues), aphospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

[0224] One skilled in the relevant art knows well how to generateoligonucleotides containing the above-described modifications. Thepresent invention is not limited to the antisense oligonucleotidesdescribed above. Any suitable modification or substitution may beutilized.

[0225] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds that are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of the presentinvention, are antisense compounds, particularly oligonucleotides, whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNaseH is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0226] Chimeric antisense compounds of the present invention may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above.

[0227] The present invention also includes pharmaceutical compositionsand formulations that include the antisense compounds of the presentinvention as described below.

[0228] B. Genetic Therapies

[0229] The present invention contemplates the use of any geneticmanipulation for use in modulating the expression of HIP1. Examples ofgenetic manipulation include, but are not limited to, gene knockout(e.g., removing the HIP1 from the chromosome using, for example,recombination), expression of antisense constructs with or withoutinducible promoters, and the like. In other embodiments, geneticmanipulation is used to deliver the HIP1 ENTH deletion mutants (e.g., tocancer cells). Delivery of nucleic acid construct to cells in vitro orin vivo may be conducted using any suitable method. A suitable method isone that introduces the nucleic acid construct into the cell such thatthe desired event occurs (e.g., expression of an antisense construct).

[0230] Introduction of molecules carrying genetic information into cellsis achieved by any of various methods including, but not limited to,directed injection of naked DNA constructs, bombardment with goldparticles loaded with said constructs, and macromolecule mediated genetransfer using, for example, liposomes, biopolymers, and the like.Preferred methods use gene delivery vehicles derived from viruses,including, but not limited to, adenoviruses, retroviruses, vacciniaviruses, and adeno-associated viruses. Because of the higher efficiencyas compared to retroviruses, vectors derived from adenoviruses are thepreferred gene delivery vehicles for transferring nucleic acid moleculesinto host cells in vivo. Adenoviral vectors have been shown to providevery efficient in vivo gene transfer into a variety of solid tumors inanimal models and into human solid tumor xenografts in immune-deficientmice. Examples of adenoviral vectors and methods for gene transfer aredescribed in PCT publications WO 00/12738 and WO 00/09675 and U.S.patent application Ser. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132,5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and5,824,544, each of which is herein incorporated by reference in itsentirety.

[0231] Vectors may be administered to subject in a variety of ways. Forexample, in some embodiments of the present invention, vectors areadministered into tumors or tissue associated with tumors using directinjection. In other embodiments, administration is via the blood orlymphatic circulation (See e.g., PCT publication 99/02685 hereinincorporated by reference in its entirety). Exemplary dose levels ofadenoviral vector are preferably 10⁸ to 10¹¹ vector particles added tothe perfusate.

[0232] C. Antibody Therapy

[0233] In some embodiments, the present invention provides antibodiesthat target HIP1 expressing tumors. In preferred embodiments, theantibodies used for cancer therapy are humanized antibodies.

[0234] In some embodiments, the therapeutic antibodies comprise anantibody generated against HIP1, wherein the antibody is conjugated to acytotoxic agent. In such embodiments, a tumor specific therapeutic agentis generated that does not target normal cells, thus reducing many ofthe detrimental side effects of traditional chemotherapy. For certainapplications, it is envisioned that the therapeutic agents will bepharmacologic agents that will serve as useful agents for attachment toantibodies, particularly cytotoxic or otherwise anticellular agentshaving the ability to kill or suppress the growth or cell division ofendothelial cells. The present invention contemplates the use of anypharmacologic agent that can be conjugated to an antibody, and deliveredin active form. Exemplary anticellular agents include chemotherapeuticagents, radioisotopes, and cytotoxins. The therapeutic antibodies of thepresent invention may include a variety of cytotoxic moieties, includingbut not limited to, radioactive isotopes (e.g., iodine-131, iodine-123,technicium-99m, indium-111, rhenium-188, rhenium-186, gallium-67,copper-67, yttrium-90, iodine-125 or astatine-211), hormones such as asteroid, antimetabolites such as cytosines (e.g., arabinoside,fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycinC), vinca alkaloids (e.g., demecolcine; etoposide; mithramycin), andantitumor alkylating agent such as chlorambucil or melphalan. Otherembodiments may include agents such as a coagulant, a cytokine, growthfactor, bacterial endotoxin or the lipid A moiety of bacterialendotoxin. For example, in some embodiments, therapeutic agents willinclude plant-, fungus- or bacteria-derived toxin, such as an A chaintoxins, a ribosome inactivating protein, α-sarcin, aspergillin,restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin,to mention just a few examples. In some preferred embodiments,deglycosylated ricin A chain is utilized.

[0235] In any event, it is proposed that agents such as these may, ifdesired, be successfully conjugated to an antibody, in a manner thatwill allow their targeting, internalization, release or presentation toblood components at the site of the targeted tumor cells as requiredusing known conjugation technology (See, e.g., Ghose et al., MethodsEnzymol., 93:280 [1983]).

[0236] For example, in some embodiments the present invention providesimmunotoxins targeted HIP1. Immunotoxins are conjugates of a specifictargeting agent typically a tumor-directed antibody or fragment, with acytotoxic agent, such as a toxin moiety. The targeting agent directs thetoxin to, and thereby selectively kills, cells carrying the targetedantigen. In some embodiments, therapeutic antibodies employ crosslinkersthat provide high in vivo stability (Thorpe et al., Cancer Res., 48:6396[1988]).

[0237] In other embodiments, particularly those involving treatment ofsolid tumors, antibodies are designed to have a cytotoxic or otherwiseanticellular effect against the tumor vasculature, by suppressing thegrowth or cell division of the vascular endothelial cells. This attackis intended to lead to a tumor-localized vascular collapse, deprivingthe tumor cells, particularly those tumor cells distal of thevasculature, of oxygen and nutrients, ultimately leading to cell deathand tumor necrosis.

[0238] In preferred embodiments, antibody based therapeutics areformulated as pharmaceutical compositions as described below. Inpreferred embodiments, administration of an antibody composition of thepresent invention results in a measurable decrease in cancer (e.g.,decrease or elimination of tumor).

[0239] D. Small Molecule Drugs

[0240] In some embodiments, the present invention provides drugs (e.g.,small molecule drugs) that reduce or eliminate cancer by inhibiting thebiological activity of HIP1. In some embodiments, small molecule drugsare identified using the drug screening methods described above. In someembodiments, the small molecule drugs are lipid analogues. In somepreferred embodiments, the small molecule drugs mimic the cell deathinducing effect of the ENTH deletion mutant of HIP1. In particularlypreferred embodiments, the small molecule drugs of the present inventionresult in the cell death of cancer, but not normal cells. In someembodiments, small molecule drugs are identified using the drug screensdescribed herein (e.g., in Example 5 and Section III above).

[0241] E. Pharmaceutical Compositions

[0242] The present invention further provides pharmaceuticalcompositions (e.g., comprising the therapeutic compounds describedabove). The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0243] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

[0244] Compositions and formulations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids or binders may be desirable.

[0245] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0246] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0247] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0248] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0249] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

[0250] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), also enhancethe cellular uptake of oligonucleotides.

[0251] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions. Thus, for example, the compositions may containadditional, compatible, pharmaceutically-active materials such as, forexample, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the compositions of thepresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention. The formulations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings and/or aromatic substances andthe like which do not deleteriously interact with the nucleic acid(s) ofthe formulation.

[0252] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents that function by a non-antisensemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. Other non-antisense chemotherapeutic agents are also withinthe scope of this invention. Two or more combined compounds may be usedtogether or sequentially.

[0253] Dosing is dependent on severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. The administering physician can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models or based on the examples describedherein. In general, dosage is from 0.01 μg to 100 g per kg of bodyweight, and may be given once or more daily, weekly, monthly or yearly.The treating physician can estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues. Following successful treatment, it may be desirable to havethe subject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years.

[0254] V. Transgenic Animals Expressing HIP1

[0255] The present invention contemplates the generation of transgenicanimals comprising an exogenous HIP1 gene or mutants and variantsthereof (e.g., truncations, deletions, insertions, or single nucleotidepolymorphisms). In other embodiments, the present invention providestransgenic animals with a knock-out of the HIP1 gene. In still furtherembodiments, transgenic animals overexpress HIP1 in specific tissues(e.g., breast, colon, and prostate). In preferred embodiments, thetransgenic animal displays an altered phenotype (e.g., increased ordecreased presence of HIP1) as compared to wild-type animals. Exemplarytransgenic animals are described in Example 4. Methods for analyzing thepresence or absence of such phenotypes include but are not limited to,those disclosed herein. In some preferred embodiments, the transgenicanimals further display an increased or decreased growth of tumors orevidence of cancer.

[0256] The transgenic animals of the present invention find use in drug(e.g., cancer therapy) screens. In some embodiments, test compounds(e.g., a drug that is suspected of being useful to treat cancer) andcontrol compounds (e.g., a placebo) are administered to the transgenicanimals and the control animals and the effects evaluated.

[0257] The transgenic animals can be generated via a variety of methods.In some embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter that allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

[0258] In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Stewart, et al,EMBO J., 6:383 [1987]). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founderswill be mosaic for the transgene since incorporation occurs only in asubset of cells that form the transgenic animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into thegermline, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo (Jahner et al., supra [1982]).Additional means of using retroviruses or retroviral vectors to createtransgenic animals known to the art involve the micro-injection ofretroviral particles or mitomycin C-treated cells producing retrovirusinto the perivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990], and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [1995]).

[0259] In other embodiments, the transgene is introduced into embryonicstem cells and the transfected stem cells are utilized to form anembryo. ES cells are obtained by culturing pre-implantation embryos invitro under appropriate conditions (Evans et al., Nature 292:154 [1981];Bradley et al., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci.USA 83:9065 [1986]; and Robertson et al., Nature 322:445 [1986]).Transgenes can be efficiently introduced into the ES cells by DNAtransfection by a variety of methods known to the art including calciumphosphate co-precipitation, protoplast or spheroplast fusion,lipofection and DEAE-dextran-mediated transfection. Transgenes may alsobe introduced into ES cells by retrovirus-mediated transduction or bymicro-injection. Such transfected ES cells can thereafter colonize anembryo following their introduction into the blastocoel of ablastocyst-stage embryo and contribute to the germ line of the resultingchimeric animal (for review, See, Jaenisch, Science 240:1468 [1988]).Prior to the introduction of transfected ES cells into the blastocoel,the transfected ES cells may be subjected to various selection protocolsto enrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

[0260] In still other embodiments, homologous recombination is utilizedknock-out gene function or create deletion mutants (e.g., truncationmutants). Methods for homologous recombination are described in U.S.Pat. No. 5,614,396, incorporated herein by reference.

EXPERIMENTAL

[0261] The following examples are provided in order to demonstrate andfurther illustrate certain preferred embodiments and aspects of thepresent invention and are not to be construed as limiting the scopethereof.

[0262] In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); and ° C. (degrees Centigrade).

EXAMPLE 1 HIP1 Expression in Cancerous and Non-Cancerous Tissues

[0263] A. Monoclonal Antibodies

[0264] The antibodies to HIP1 were generated by isolating a human HIP1cDNA and creating a bacterial expression vector containing the sequencesencoding the carboxyl half of the HIP1 protein. The expressed andpurified recombinant protein was use to immunize mice and createmonoclonal antibodies by standard methods. The specificity of theantibodies to HIP1 was confirmed by comparing Western blot analyses ofmurine embryonic fibroblast extracts derived from embryos with HIP1deleted and their wild type litter mates (data not shown). Threemonoclonal antibodies were found to be useful for Western blot analysisof HIP1, as well as for IHC of human tissue. These antibodies weredesignated HIP1-4B10, HIP1-1A1 and HIP1-1B11. Antibodies 4B10 and 1A1recognize only human HIP1. Antibody 1B11 recognizes both human and mouseHIP1. The hybridomas producing antibodies HIP1-4B10, 1A1 and HIP1-1B11were deposited with ATCC under numbers pending.

[0265] B. Tissue Microarrays

[0266] A high-density tissue microarray of multiple primary tumors wasobtained from the National Cancer Institute. High-density tissuemircroarrays for prostate tumors were assembled as previously described(Perrone et al., J. Natl. Cancer Inst., 92:937 [2000]). Cases ofclinically localized prostate cancer were from the University ofMichigan Prostate SPORE tumor bank. The advanced prostate tumors werecollected from a series of rapid autopsies performed at the Universityof Michigan on men who died of metastatic prostate cancer. Autopsieswere performed 4-6 h after death.

[0267] C. Immunohistochemistry

[0268] Standard biotin-avidin-complex IHC was performed. Immunostainingintensity was scored as absent, weak, moderate or high. In order toqualify as high, staining intensity had to be equivalent to thatobserved in endothelia of small blood vessels of the tissue. There wasno staining when secondary antibody was used alone as a negativecontrol.

[0269] D. Results

[0270] 1. HIP1 in Cancer Cell Lines

[0271] Western blotting of extracts from the 60 cancer cell lines of theNCI's in vitro anti-cancer drug screen (Monks et al., J. Natl. Cancerinst., 83:757 [1991]) was used to investigate the expression of HIP1 invarious cancers. Protein extracts (50 μg) derived from the 60 cell lineswere separated on a 6% polyacrylamide gel, transferred tonitrocellulose, and blotted with a mix of the anti-HIP1 monoclonalantibodies 4B10 and 1B11.

[0272] The results demonstrated that HIP1 was highly expressed in mostof the cancer cell lines (50/53) derived from solid tumors (FIG. 1). Thecancers represented include colon, breast, melanoma, ovary, prostate,kidney and lung. HIP1 was expressed at low levels or was absent in celllines derived from hematologic malignancies. The exception was the highlevel of HIP1 expression in the K562 cell line, which is derived from apatient with chronic myelogenous leukemia. This leukemia, when in thechronic phase, is a myeloproliferative syndrome similar to the CMML thatcan be caused by expression of the HIP1/PDGFβR tyrosine kinase (Ross etal., Blood 91:4419 [1998]).

[0273] In order to further investigate the differential expression ofHIP1, tissue microarrays were used to examine HIP1 expression in largenumbers of primary tumor specimens. Microarray slides were subjected toimmunohistochemical analysis with the anti-HIP1 monoclonal antibody4B10. Using the multi-tumor tissue microarray from the National CancerInstitute designated TARP1 (Perrone et al., J. Natl. Cancer Inst.,92:937 [2000]), HIP1 was found to be moderately-to-highly expressed in alarge percentage of CNS, breast, colon, lung, melanoma, ovarian andprostate cancers (FIG. 2). The highest levels of expression were foundin ovarian cancer and melanoma. As predicted from the lack of expressionof HIP1 in the hematologically derived NCI-ACDS cancer cell lines, therewas no detectable HIP1 expression in over 80% of primary lymphomasamples.

[0274] 2. HIP1 Expression in Normal Tissues

[0275] Next, IHC for HIP1 expression was examined in multiple normaltissues. HIP1 was expressed most highly in the endothelium of smallblood vessels. HIP1 was also highly expressed in several secretoryepithelia, including breast ductal epithelium, gastric epithelium andkidney distal tubular epithelium. Other tissues that expressed HIP1 athigh levels included the lung epithelia, heart muscle, choroid plexus ofthe brain, various peripheral nervous system ganglia and liver. HIP1 isalso expressed in the post-meiotic spermatids of the seminiferoustubules of the testis. The epithelium of the colon and prostate did notdetectably express HIP1.

[0276] 3. HIP1 Expression in Colon and Prostate Tissues

[0277] To study the expression of HIP1 in tumors where its expressionseemed to be limited to the neoplastic epithelium, anti-HIP1 monoclonalantibodies were first used to stain colon cancers. Standard slides fromarchival tissue samples were obtained from 25 patients withwell-differentiated colon cancers. In separate experiments, slides werestained with the monoclonal anti-HIP1 antibodies 4B10 and 1B11.

[0278]FIG. 3a shows a histogram of HIP1 expression in normal tissue andwell differentiated colon cancer tissue. On staining with either of theanti-HIP1 antibodies (4B10 or 1B11), benign tissue was found to notexpress HIP1, while adjacent colon tumors had high levels of HIP1expression. Quantitatively, benign epithelium of the colon never hadmoderate or high expression, whereas 48% of the well differentiatedcolon tumors had moderate or high HIP1 expression (FIG. 3a). 84% ofbenign colon epithelium samples did not express HIP1 compared to 8% ofthe colon tumor samples.

[0279] In addition to colon cancer, prostate cancer demonstratedmoderate to high levels of HIP1 expression (FIG. 2h), while benignepithelia did not express HIP1. To study the aberrant HIP1 expression inmore detail, three prostate tissue microarrays containing a total of 853tissue spots from 114 patients with localized prostate cancer werestained with the HIP1 monoclonal antibodies. These samples includedbenign prostate tissue, prostatic intraepithelial neoplasia (PIN) andprostate cancer (PCa). In addition, a separate tissue microarray thatcontained 135 tumor samples from 14 patients with hormone-refractorymetastatic PCa was examined for HIP1 expression. As in the colon,striking differences were noted in staining between neoplastic andbenign epithelia. PCA from the prostate tissue microarray demonstratedhigh HIP1 expression juxtaposed to non-HIP1 expressing benign prostaticepithelium. 95% of normal epithelium samples did not express HIP1. Theremaining 5% had weak levels of expression and there was no moderate orhigh expression in any of the normal epithelia examined. Amongneoplastic lesions, the precursor of prostate cancer, PIN, had thelowest number of samples with moderate or strong levels of HIP1expression (25% of the 230 samples). Localized PCa had an intermediatenumber (51% of the 463 samples) and metastatic prostate cancer had thehighest number of samples expressing moderate to strong levels of HIP1(70% of the 135 samples). This difference in levels of HIP1 inprogressively more advanced PCA was statistically significant (FIG. 3b,Pearson's chi-squared, P<0.0001).

[0280] 4. Effect of HIP1 Expression on Clinical Outcome in PCA

[0281] The above experiments demonstrated that there were significantnumbers of samples of colon cancer, PIN and PCA that did not expressHIP1, similar to normal epithelia, which lacks HIP1 expression. Thepresent invention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, since incrementally more malignant cancers hadhigher frequencies of HIP1 expression, it is hypothesized that differentlevels of HIP1 expression predict different behaviors in tumors. One wayto look at these behaviors is to examine clinical outcomes.

[0282] Because PCA is a histologically heterogeneous tumor, an algorithmwas devised for relating levels of HIP1 expression in multiple tumorspecimens from each patient to an overall score for each patient.Because of heterogeneity of PCA and to evaluate the samples in the leastbiased way possible the following algorithm was developed. Heterogeneitywas reflected in the PCA microarrays by the fact that each patient withPCa had multiple microarray samples (average of 4 per patient) that didnot display the same levels of HIP1 staining (FIG. 4). The highest levelof HIP1 expression from multiple tumor specimens was therefore used torepresent the overall patient score. Next, a dichotomous relationshipwas defined, where each patient received either a “zero” or a “one” forabsent expression versus weak, moderate or high expression,respectively. Any cases where there was only a single specimen availablewere excluded from analysis.

[0283] A tissue microarray that was derived from prostatic tumorssurgically resected from 121 patients, as well as a tissue microarraythat was derived from hormone-refractory metastatic PCa from 15 patientswere used. FIG. 4a shows an analysis of HIP1 expression in individualpatients reveals that there were progressively higher frequencies ofHIP1 expression in benign (n=89), PIN (n=89), PCa (n=114), andmetastatic cases (n=14). Conversely, there were progressively lowerfrequencies of the lack of HIP1 expression among the same. Thesedifferences are statistically significant (Pearson's chi-square 2;p<0.001), and the presence of HIP1 expression correlated significantlywith the ordinal categories of benign vs. PIN vs. PCa vs. Metastatic(Spearman's correlation coefficient, 0.664, p<0.001).

[0284] An outcomes analysis was next performed on 463 samples oflocalized PCA from 114 patients represented by the third bar of eachgroup of patients in FIG. 4a. To avoid introduction of bias, themicroarrays were scored for HIP1 expression before clinical data wasrevealed to the readers of the microarrays. Taking the highest score ofall the samples from each patient, Pearson's chi-square was used todetermine if there were clinical implications associated with HIP1expression. Various clinical parameters such as Gleason score, tumorsize, seminal vesicle invasion, extra-prostatic extension, and relapsefrom PCa were evaluated. Men who have elevated PSA levels subsequent toradical prostatectomy die because of recurrent prostate cancer. This istermed PSA recurrence. Patients whose tumors did not stain for HIP1expression did not develop a PSA recurrence (Table 1). In comparison apreoperative PSA of <4 (normal) compared to PSA>4 was also a significantgood prognostic factor (Pearson's chi-squared; P<0.034). The ability ofa HIP1 negative tumor to predict recurrence free survival, also termednegative predictive value was 100% while PSA<4 was 95.6% predictive.Table 2 demonstrates that HIP1 expression is independent of traditionalhistopathologic indicators as a molecular predictor of relapse fromprostate cancer

[0285] The survival advantage of PCa patients with tumors that had noHIP1 expression was demonstrated by the Kaplan-Meier survival curveshown in FIG. 4b. The curve compares patients with prostate confinedtumors that did not express HIP1 (open circles) with those patientswhose tumors did express HIP1 (open triangles). Again, the same group of114 patients with prostate confined tumors was used in this analysis asin Table 1. All the patients that lacked HIP1 expression survived 67months without evidence cf recurrence. In contrast, 28% of the patientswhose tumors expressed HIP1 died of prostate cancer. These data,together with the correlation of HIP1 expression with both metastatictumors and overall death from prostate cancer suggest that HIP1 predictsboth invasiveness as well as metastatic potential of prostate cancer.

[0286] In summary, this example describes the ability of HIP1 expressionto mark colon and prostate tumors. The level of expression of HIP1 inprostate tumors was evaluated in detail and correlated with progressionof prostate cancer. This was evidenced by a lack of expression in thebenign epithelium compared to the highest levels of expression in themost deadly cases of metastatic PCa. In addition, there was a survivaladvantage in patients whose prostate confined PCA's did not express HIP1compared to patients whose tumors did express HIP1. TABLE 1 HIP1expression and preoperative PSA as predictors of PSA recurrence No PSArecur PSA recur Pearson's Two-sided Significance (n, %) (n, %) X² p HIP1Absent 14 (100%)  0 (0%) 4.034 0.045 HIP1 Expressed 77 (77%) 23 (23%)Preoperative 22 (95.7%)  1 (4.3%) 4.482 0.034 PSA <4 Preoperative 69(75.8%) 22 (24.1%) PSA >4

[0287] TABLE 2 HIP1 expression is independent of traditionalhistopathologic indicators as a molecular predictor of relapse fromprostate cancer Tumor HIP1 Staining Negative Positive Significance¹ (no.of patients) (no. of patients) (two-tailed p) Gleason <7 9 35 0.038Score² >7 5 65 Local No 13 70 0.044 Spread³ Yes 1 30 PSA re- No 14 770.009 currence⁴ Yes 0 23

EXAMPLE 2 HIP1 Expression in a Mouse Model of Prostate Cancer

[0288] This example describes an investigation of the expression of HIP1in a transgeric mouse model of prostate cancer, designated the TRAMPmouse (Greenberg et al., PNAS 92:3439 [1995]). The TRAMP mouse wascreated by use of a transgene where the rat probasin promoter drivesexpression of SV40 early genes (T and t; Tag). SV40 Tag interacts withp53 and the retinoblasoma gene product Rb abrogating their tumorsuppressor gene functions.

[0289] HIP1 was found to be overexpressed in 50% of prostate tumors fromTRAMP mice (n=10) compared to the normal prostate (FIG. 6a), suggestingthat its expression is an active component of tumorigenesis in thesemice. The heterogeneity of HIP1 expression is similar to that seen inhuman prostate cancers.

EXAMPLE 3 HIP1 ENTH Mutant

[0290] The effect of expression of a HIP1 dominant negative mutant oncellular survival was next investigated. At the molecular level, thedominant negative mutant of HIP1 (HIP1/deltaE lacking the ENTH domain)promoted cell death but expression of wild type HIP1 (FLHIP1) or vectoralone (pcDNA3) did not induce cell death (FIG. 6b). 39% ofHIP1/DE-expressing cells and 50% of caspase 9-expressing cells wereapoptotic. FIG. 12 shows the correction of HIP1 deltaE apoptosis by theexpression fall length HIP1 (FLPHIP1). FIG. 13 shows the correction ofapoptosis by the expression of dominant negative caspase 9 (DN Casp 9)and constitutatively active Akt (Myr-AKT) but not dominant negativecaspase 8 (DN I-FLICE) or activated ras (Ras-V12). Expression of theendogenous HIP1, wild type HIP1, HIP1/DE and Caspase 9 was documented byWestern blot analysis as shown in the bottom of FIG. 6b.

EXAMPLE 4 HIP1 Transgenic Mice

[0291] This Example describes the construction of several HIP1transgenic mice lines.

[0292] A. Generation of HIP1-Knockout Mice

[0293] This Example describes the inactivation of HIP1 by thereplacement of exons 2 to 7 with a neomycin resistance gene (neo^(r)).The HIP1 knockout vector (pHIP1KO) was constructed from two mousegenomic clones. Mouse genomic clones were obtained using informationfrom the human genomic structure deduced from the human genomic sequenceof the long arm of chromosome 7 (GenBank accession no. AC004491) and thecDNA sequence (Genbank accession no. HSHIP1PRO). The mouse genomicclones were isolated from a 129/Sv genomic BAC library for subcloningand restriction mapping. The mouse cDNA used to screen for the genomicclones was obtained by homology screening from a fetal mouse cDNAlibrary (provided by Lewis Chodosh, University of Pennsylvania). ThiscDNA was also used to deduce the 5′ end of the HIP1 cDNA gene andpromoter region. A restriction map of the 5′ end of the gene as one ofthe HIP1 BAC clones contained exon 2 and the other contained exons 3 to8. Using these two clones, the targeting vector, pHIP1KO, including theknockout region of approximately 13.7 kb (including exons 2 to 7), wascompleted. The most 5′ HIP1 subclone (subclone EcoB/E) was digested withXbaI, filled in, and digested with XhoI. The resultant 3.5-kb fragmentcontaining intronic sequence 500 bp upstream of the 5′ arm was clonedinto the NheI (blunted)-XhoI site of 38LoxPNeo (a modified version ofpGT-N38 from New England Biolabs) that has a LoxpNeo cassette clonedinto the polylinker. As a result, the 3.5-kb 5′ arm was just 5′ of theLoxPNeo cassette. This intermediate vector was then digested with XbaIlocated 3′ to the Neo cassette and blunted. A subclone (H/B) thatcontained exon 8 and flanking 5′ and 3′ intronic sequences was digestedwith HindIII (the 4.0-kb 3′ arm), which released the entire 4 kb of thissubclone. This HindIII fragment was then blunted and ligated with theXbaI (blunted) intermediate vector. The 5′ junction of this resultantvector was sequenced to determine if the 4-kb 3′ arm was in the correctorientation. The final targeting vector was electroporated into 129SvJRW1 ES cells (Incyte Genomics, St. Louis, Mo.), selected for G418resistance, and screened by Southern blotting for correctly targetedclones. Generation of chimeric mice and germline transmission of themutant allele were achieved using standard techniques. Briefly, Thecorrectly targeted ES cell clone was injected into C57BL/6 blastocysts.Chimeras with a high percentage of agouti were mated to C57BL/6 females,and F1 agouti pups were genotyped by Southern blotting. An equaldistribution of wild-type and heterozygous mice among the agouti F1animals was found (31_/− to 30_/_ animals). F1 heterozygotes weresubsequently intercrossed to generate F2 animals, which were genotypedat 3 weeks of age.

[0294] Tail biopsies of 3-week-old mice were performed at weaning.Genomic DNA was isolated using the Promega Wizard kit as specified bythe manufacturer digested with EcoRI overnight, and run on a 0.7%agarose-Tris-borate EDTΔ (TBE) gel to separate 16.5-kb (wild type) and12.0-kb (recombinant) bands detected with the 5′ probe. The gel was thenblotted onto Hybond-N filter (Amersham-Pharmacia) and blocked in 20 μgof salmon sperm DNA per ml in hybridization buffer (Amersham-Pharmacia)for 3 h at 65° C. 32 P labeling of the 5′ probe was done byrandom-primed labeling (Roche) with [32 P]dCTP (NEN). The blots werethen hybridized for 14 to 20 h at 65° C., washed twice in 2× SSC (1× SSCis 0.15 M NaCl plus 0.015 M sodium citrate) for 20 min and twice in 1×SSC for 10 min, and imaged on Kodak Biomax film.

[0295] During the construction of the targeting vector to knock outHIP1, a human HIP1 cDNA clone was used to screen a mouse embryonic cDNAlibrary and a 5′ murine HIP1 clone was isolated. This CDNA clone carrieda new exon not previously described. By homology searching, ahigh-probability match with a 161-bp region of human chromosome 7q11(BAC clone CTB-139P11) was identified (FIG. 18). The first intronseparating this newly identified first exon and the second exon spans139.5 kb. Exon 2 was previously designated exon 1 (Kalchman et al., Nat.Gent. 16:44 [1997]). Unlike exon 2, the newly discovered exon 1 containsa cluster of three in-frame ATG sequences, at +41, +50, and +62 in exon1, with strong Kozak consensus sequences. Initiation of translation fromthe first ATG sequence would result in a HIP1 protein of 116 kDa,consistent with the size observed by Western blot analysis. Examinationof the genomic sequence from BAC clone CTB-139P11 demonstrates aputative promoter region in the 5′-flanking sequence of the open readingframe that begins with HIP1 exon 1. This region contains a CCAAT box at−71 (FIG. 18) of exon 1 as well as several GC-rich areas correspondingto GC boxes (FIG. 18). Furthermore, several putative binding sites fortranscription factors were found in this 5′-flanking region, includingthose for NF-κB, EGR-1, and c-myb. The predicted protein sequence forexon 1 encodes an additional 42 amino acids, compared with the sequencerecorded at the National Center for Biotechnology Information (accessionno. XP 004910). Analysis of the predicted protein sequence for fulllength HIP1 demonstrates a putative P-4,5-P2 and PI-3,4,5-P3 bindingENTH motif, in addition to putative clathrin and AP2 binding sequences,a leucine zipper, and a TALIN homology domain. The leucine zipper lieswithin a region that demonstrates homology to the central rod region ofintermediate filament proteins.

[0296] B. HIP1 Knock-In

[0297] An additional transgenic mouse was constructed that had the HIP1gene inactivated by a knock-in of exogenous DNA. FIG. 15 gives anoverview of the construction of the vector. The vector (HIP1/PDGFβKI)was constructed by:

[0298] 1. resequence 38LoxPNeo/HIP1 construct (See Section A above) withNeoR, choose sequence outside XhoI site to go 3′ (primer 1). Primer 2 isfrom human cDNA, the same as 156R. Primer 3 is from human cDNA 157F,5′-GCT GTA AAG GAA AAA CAC GCC-3′ (SEQ ID NO: 5). Primer 4 is from humancDNA, the same as 588R.

[0299] 2. Perform PCR reaction: Template: subclone EcoB/E; Primers: (1)and (2)

[0300] Blunt-end PCR fragment˜2.5 kb

[0301] Digest with XhoI

[0302] 3. Perform PCR reaction: Template: pcDNA3.HIP/P; Primers: (3) and(4)

[0303] Blunt-end PCR fragment˜0.4 kb

[0304] 4. Digest ASIL vector with SacI+AscI, elute˜2.1 kb bGHpA-loxPNeofragment

[0305] Digest pGL-1 with SacI+AscI

[0306] Ligate

[0307] Obtain plasmid pGL-1/step4, insert˜2.1 kb

[0308] 5. Three fragment ligation: pGL-1/Step4 digested withSalI+Ecl136I

[0309] PCR fragment from step 2

[0310] PCR fragment form step 3

[0311] Obtain plasmid pGL-1/step5, insert˜5.0 kb

[0312] 6. Digest pcDNA3.HIP/P with NotI-blunt end+HpaI, elute˜3.6 kbfragment

[0313] Digest plasmid pGL-1/step with HpaI

[0314] Ligate

[0315] Obtain plasmid pGL-1/step6, insert˜8.6 kb

[0316] 7. Digest pGL-1/step6 with NotI+AscI, blunt-end, elute˜8.6 kbfragment

[0317] Digest 38LoxPNeo/HIP1 with XhoI+SfiI, blunt-end

[0318] Ligate

[0319] Obtain KI vector, total size˜18.9 kb

[0320] Embryonic stem (ES) cells were electroporated and screened forcorrect targeting using two DNA probes, one complementary to a 5′ regionand one complementary to a 3′ region. DNA was digested with restrictionenzymes and probed. Wild-type and mutant give different sizedrestriction fragments that were identified with the probes. One of theES cells had a 248 bp deletion of parts of exon 2 and intron 1 (FIG.19). Transgenic mice were also generated from the construct.

[0321] Targeted ES cells were then injected into blastocysts that werethen impregnated into pseudopregnant females. The resultant chimeraswere then bred to make F1s. The F1 generation is crossed onto variouscre backgrounds.

[0322] C. Conditional Knock-Out Mouse

[0323] The Example describes the construction of a conditional HIP1knock-out transgenic mouse. Infection of the mouse with an adenoviralvector expressing the cre gene will acutely decrease the levels of HIP1via recombination. An overview of the cloning strategy is described inFIG. 16. The cloning strategy was as follows:

[0324] 1. Clone 3.2 kb KpnI-BamHI fragment (5′ homologous arm) into98TB/KpnI+BamHI digestion.

[0325] Obtain plasmid Step1˜5.2 kb.

[0326] 2. Clone 3.5 kb EcoRV-HindIII fragment (3′ homologous arm) intoplasmid Step 1/EcoRV+HindIII digestion.

[0327] Obtain plasmid Step2˜8.7 kb.

[0328] 3. Clone 5.0 kb BamHI (blunt end)-EcoRV fragment (conditional KOregion) into 38LoxPNeo/HindIII (blunt end) digestion.

[0329] Screen for correct orientation.

[0330] Obtain plasmid Step3˜9.7 kb.

[0331] 4. Clone 6.8 kb XhoI (blunt end)-SfiI (blunt end) fragment(conditional KO-Neo) from plasmid Step3 into plasmid Step2/AscI (bluntend) digestion.

[0332] Screen for correct orientation.

[0333] Obtain conditional KO vector˜15.5 kb.

[0334] Transgenic mice were generated as described in section B above.

EXAMPLE 5 Drug Screening

[0335] This Example describes exemplary drug screening methods suitablefor screening compounds for HIP1 inhibitory activity. A high throughputscreening method is utilized.

[0336] A. Embryonic Fibroblast Screening

[0337] HIP1+embryonic fibroblasts are obtained from wild-type mice andHIP1−/−embryonic fibroblasts are obtained from HIP1 knockout mice (Raoet al., Mol Cell Biol 21:7796 [2001]). These fibroblasts are culturedand libraries of therapeutic compounds are added to the cultures.Specific inhibitors of HIP1 are then identified as compounds that killthe HIP1+fibroblasts but not the HIP1−fibroblasts.

[0338] B. Cancer Cell Line Screeing

[0339] Drug screens are also performed using HIP1 expressing cell linesand HIP1 non-expressing colo205 cells (ATCC, obtained from a patientwith metastatic colon cancer). The two cell lines are cultured in thepresence of a library of therapeutic compounds. Compounds are identifiedthat kill HIP1+ but not HIP1−cell lines.

1 6 1 4534 DNA Homo sapiens misc_feature (4521)..(4521) The nucleotide“n” can be either a,t,c, or g 1 ccaagcttgg tacccccggg gcagccgagggcccctgact cggctcctcg cggcgacatg 60 gatcggatgg ccagctccat gaagcaggtgcccaacccac tgcccaaggt gctgagccgg 120 cgcggggtcg gcgctgggct ggaggcggcggagcgcgaga gcttcgagcg gactcagact 180 gtcagcatca ataaggccat taatacgcaggaagtggctg taaaggaaaa acacgccaga 240 acgtgcatac tgggcaccca ccatgagaaaggggcacaga ccttctggtc tgttgtcaac 300 cgcctgcctc tgtctagcaa cgcagtgctctgctggaagt tctgccatgt gttccacaaa 360 ctcctccgag atggacaccc gaacgtcctgaaggactctc tgagatacag aaatgaattg 420 agtgacatga gcaggatgtg gggctacctgagcgaggggt atggccagct gtgcagcatc 480 tacctgaaac tgctaagaac caagatggagtaccacacca aaaatcccag gttcccaggc 540 aacctgcaga tgagtgaccg ccagctggacgaggctggag aaagtgacgt gaacaacttt 600 tcccagttaa cagtggagat gtttgactacctggagtgtg aactcaacct cttccaaaca 660 gtattcaact ccctggacat gtcccgctctgtgtccgtga cggcagcagg gcagtgccgc 720 ctcgccccgc tgatccaggt catcttggactgcagccacc tttatgacta cactgtcaag 780 cttctcttca aactccactc ctgcctcccagctgacaccc tgcaaggcca ccgggaccgc 840 ttcatggagc agtttacaaa gttgaaagatctgttctacc gctccagcaa cctgcagtac 900 ttcaagcggc tcattcagat cccccagctgcctgagaacc cacccaactt cctgcgagcc 960 tcagccctgt cagaacatat cagccctgtggtggtgatcc ctgcagaggc ctcatccccc 1020 gacagcgagc cagtcctaga gaaggatgacctcatggaca tggatgcctc tcagcagaat 1080 ttatttgaca acaagtttga tgacatctttggcagttcat tcagcagtga tcccttcaat 1140 ttcaacagtc aaaatggtgt gaacaaggatgagaaggacc acttaattga gcgactatac 1200 agagagatca gtggattgaa ggcacagctagaaaacatga agactgagag ccagcgggtt 1260 gtgctgcagc tgaagggcca cgtcagcgagctggaagcag atctggccga gcagcagcac 1320 ctgcggcagc aggcggccga cgactgtgaattcctgcggg cagaactgga cgagctcagg 1380 aggcagcggg aggacaccga gaaggctcagcggagcctgt ctgagataga aaggaaagct 1440 caagccaatg aacagcgata tagcaagctaaaggagaagt acagcgagct ggttcagaac 1500 cacgctgacc tgctgcggaa gaatgcagaggtgaccaaac aggtgtccat ggccagacaa 1560 gcccaggtag atttggaacg agagaaaaaagagctggagg attcgttgga gcgcatcagt 1620 gaccagggcc agcggaagac tcaagaacagctggaagttc tagagagctt gaagcaggaa 1680 cttgccacaa gccaacggga gcttcaggttctgcaaggca gcctggaaac ttctgcccag 1740 tcagaagcaa actgggcagc cgagttcgccgagctagaga aggagcggga cagcctggtg 1800 agtggcgcag ctcataggga ggaggaattatctgctcttc ggaaagaact gcaggacact 1860 cagctcaaac tggccagcac agaggaatctatgtgccagc ttgccaaaga ccaacgaaaa 1920 atgcttctgg tggggtccag gaaggctgcggagcaggtga tacaagacgc cctgaaccag 1980 cttgaagaac ctcctctcat cagctgcgctgggtctgcag atcacctcct ctccacggtc 2040 acatccattt ccagctgcat cgagcaactggagaaaagct ggagccagta tctggcctgc 2100 ccagaagaca tcagtggact tctccattccataaccctgc tggcccactt gaccagcgac 2160 gccattgctc atggtgccac cacctgcctcagagccccac ctgagcctgc cgactcactg 2220 accgaggcct gtaagcagta tggcagggaaaccctcgcct acctggcctc cctggaggaa 2280 gagggaagcc ttgagaatgc cgacagcacagccatgagga actgcctgag caagatcaag 2340 gccatcggcg aggagctcct gcccaggggactggacatca agcaggagga gctgggggac 2400 ctggtggaca aggagatggc ggccacttcagctgctattg aaactgccac ggccagaata 2460 gaggagatgc tcagcaaatc ccgagcaggagacacaggag tcaaattgga ggtgaatgaa 2520 aggatccttg gttgctgtac cagcctcatgcaagctattc aggtgctcat cgtggcctct 2580 aaggacctcc agagagagat tgtggagagcggcaggggta cagcatcccc taaagagttt 2640 tatgccaaga actctcgatg gacagaaggacttatctcag cctccaaggc tgtgggctgg 2700 ggagccactg tcatggtgga tgcagctgatctggtggtac aaggcagagg gaaatttgag 2760 gagctaatgg tgtgttctca tgaaattgctgctagcacag cccagcttgt ggctgcatcc 2820 aaggtgaaag ctgataagga cagccccaacctagcccagc tgcagcaggc ctctcgggga 2880 gtgaaccagg ccactgccgg cgttgtggcctcaaccattt ccggcaaatc acagatcgaa 2940 gagacagaca acatggactt ctcaagcatgacgctgacac agatcaaacg ccaagagatg 3000 gattctcagg ttagggtgct agagctagaaaatgaattgc agaaggagcg tcaaaaactg 3060 ggagagcttc ggaaaaagca ctacgagcttgctggtgttg ctgagggctg ggaagaagga 3120 acagaggcat ctccacctac actgcaagaagtggtaaccg aaaaagaata gagccaaacc 3180 aacaccccat atgtcagtgt aaatccttgttacctatctc gtgtgtgtta tttccccagc 3240 cacaggccaa atccttggag tcccaggggcagccacacca ctgccattac ccagtgccga 3300 ggacatgcat gacacttcca aagactccctccatagcgac accctttctg tttggaccca 3360 tggatttcca ctgcttctta tggtggttggttgggttttt tggttttgtt tttttttttt 3420 aagtttcact cacatagcca actctcccaaagggcacacc cctggggctg agtctccagg 3480 gccccccaac tgtggtagct ccagcgatggtgctgcccag gcctctcggt gctccatctc 3540 cgcctccaca ctgaccaagt gctggcccacccagtccatg ctccagggtc aggcggagct 3600 gctgagtgac agctttcctc aaaaagcagaaggagagtga gtgcctttcc ctcctaaagc 3660 tgaatcccgg cggaaagcct ctgtccgcctttacaaggga gaagacaaca gaaagaggga 3720 caagagggtt cacacagccc agttcccgtgacgaggctca aaaacttgat cacatgcttg 3780 aatggagctg gtgagatcaa caacactacttccctgccgg aatgaactgt ccgtgaatgg 3840 tctctgtcaa gcgggccgtc tcccttggcccagagacgga gtgtgggagt gattcccaac 3900 tcctttctgc agacgtctgc cttggcatcctcttgaatag gaagatcgtt ccaccttcta 3960 cgcaattgac aaacccggaa gatcagatgcaattgctccc atcagggaag aaccctatac 4020 ttggtttgct acccttagta tttattactaacctccctta agcagcaaca gcctacaaag 4080 agatgcttgg agcaatcaga acttcaggtgtgactctagc aaagctcatc tttctgcccg 4140 gctacatcag ccttcaagaa tcagaagaaaggccaaggtg ctggactgtt actgacttgg 4200 atcccaaagc aaggagatca tttggagctcttgggtcaga gaaaatgaga aaggacagag 4260 ccagcggctc caactccttt cagccacatgccccaggctc tcgctgccct gtggacagga 4320 tgaggacaga gggcacatga acagcttgccagggatgggc agcccaacag cacttttcct 4380 cttctagatg gaccccagca tttaagtgaccttctgatct tgggaaaaca gcgtcttcct 4440 tctttatcta tagcaactca ttggtggtagccatcaagca cttcggaatt cctgcagccc 4500 gggcggccgc tcgagcatgc nntagagggcccta 4534 2 1475 PRT Homo sapiens misc_feature (1472)..(1472) The aminoacid “X” can be any amino acid 2 Met Asp Arg Met Ala Ser Ser Met Lys GlnVal Pro Asn Pro Leu Pro 1 5 10 15 Lys Val Leu Ser Arg Arg Gly Val GlyAla Gly Leu Glu Ala Ala Glu 20 25 30 Arg Glu Ser Phe Glu Arg Thr Gln ThrVal Ser Ile Asn Lys Ala Ile 35 40 45 Asn Thr Gln Glu Val Ala Val Lys GluLys His Ala Arg Thr Cys Ile 50 55 60 Leu Gly Thr His His Glu Lys Gly AlaGln Thr Phe Trp Ser Val Val 65 70 75 80 Asn Arg Leu Pro Leu Ser Ser AsnAla Val Leu Cys Trp Lys Phe Cys 85 90 95 His Val Phe His Lys Leu Leu ArgAsp Gly His Pro Asn Val Leu Lys 100 105 110 Asp Ser Leu Arg Tyr Arg AsnGlu Leu Ser Asp Met Ser Arg Met Trp 115 120 125 Gly Tyr Leu Ser Glu GlyTyr Gly Gln Leu Cys Ser Ile Tyr Leu Lys 130 135 140 Leu Leu Arg Thr LysMet Glu Tyr His Thr Lys Asn Pro Arg Phe Pro 145 150 155 160 Gly Asn LeuGln Met Ser Asp Arg Gln Leu Asp Glu Ala Gly Glu Ser 165 170 175 Asp ValAsn Asn Phe Ser Gln Leu Thr Val Glu Met Phe Asp Tyr Leu 180 185 190 GluCys Glu Leu Asn Leu Phe Gln Thr Val Phe Asn Ser Leu Asp Met 195 200 205Ser Arg Ser Val Ser Val Thr Ala Ala Gly Gln Cys Arg Leu Ala Pro 210 215220 Leu Ile Gln Val Ile Leu Asp Cys Ser His Leu Tyr Asp Tyr Thr Val 225230 235 240 Lys Leu Leu Phe Lys Leu His Ser Cys Leu Pro Ala Asp Thr LeuGln 245 250 255 Gly His Arg Asp Arg Phe Met Glu Gln Phe Thr Lys Leu LysAsp Leu 260 265 270 Phe Tyr Arg Ser Ser Asn Leu Gln Tyr Phe Lys Arg LeuIle Gln Ile 275 280 285 Pro Gln Leu Pro Glu Asn Pro Pro Asn Phe Leu ArgAla Ser Ala Leu 290 295 300 Ser Glu His Ile Ser Pro Val Val Val Ile ProAla Glu Ala Ser Ser 305 310 315 320 Pro Asp Ser Glu Pro Val Leu Glu LysAsp Asp Leu Met Asp Met Asp 325 330 335 Ala Ser Gln Gln Asn Leu Phe AspAsn Lys Phe Asp Asp Ile Phe Gly 340 345 350 Ser Ser Phe Ser Ser Asp ProPhe Asn Phe Asn Ser Gln Asn Gly Val 355 360 365 Asn Lys Asp Glu Lys AspHis Leu Ile Glu Arg Leu Tyr Arg Glu Ile 370 375 380 Ser Gly Leu Lys AlaGln Leu Glu Asn Met Lys Thr Glu Ser Gln Arg 385 390 395 400 Val Val LeuGln Leu Lys Gly His Val Ser Glu Leu Glu Ala Asp Leu 405 410 415 Ala GluGln Gln His Leu Arg Gln Gln Ala Ala Asp Asp Cys Glu Phe 420 425 430 LeuArg Ala Glu Leu Asp Glu Leu Arg Arg Gln Arg Glu Asp Thr Glu 435 440 445Lys Ala Gln Arg Ser Leu Ser Glu Ile Glu Arg Lys Ala Gln Ala Asn 450 455460 Glu Gln Arg Tyr Ser Lys Leu Lys Glu Lys Tyr Ser Glu Leu Val Gln 465470 475 480 Asn His Ala Asp Leu Leu Arg Lys Asn Ala Glu Val Thr Lys GlnVal 485 490 495 Ser Met Ala Arg Gln Ala Gln Val Asp Leu Glu Arg Glu LysLys Glu 500 505 510 Leu Glu Asp Ser Leu Glu Arg Ile Ser Asp Gln Gly GlnArg Lys Thr 515 520 525 Gln Glu Gln Leu Glu Val Leu Glu Ser Leu Lys GlnGlu Leu Ala Thr 530 535 540 Ser Gln Arg Glu Leu Gln Val Leu Gln Gly SerLeu Glu Thr Ser Ala 545 550 555 560 Gln Ser Glu Ala Asn Trp Ala Ala GluPhe Ala Glu Leu Glu Lys Glu 565 570 575 Arg Asp Ser Leu Val Ser Gly AlaAla His Arg Glu Glu Glu Leu Ser 580 585 590 Ala Leu Arg Lys Glu Leu GlnAsp Thr Gln Leu Lys Leu Ala Ser Thr 595 600 605 Glu Glu Ser Met Cys GlnLeu Ala Lys Asp Gln Arg Lys Met Leu Leu 610 615 620 Val Gly Ser Arg LysAla Ala Glu Gln Val Ile Gln Asp Ala Leu Asn 625 630 635 640 Gln Leu GluGlu Pro Pro Leu Ile Ser Cys Ala Gly Ser Ala Asp His 645 650 655 Leu LeuSer Thr Val Thr Ser Ile Ser Ser Cys Ile Glu Gln Leu Glu 660 665 670 LysSer Trp Ser Gln Tyr Leu Ala Cys Pro Glu Asp Ile Ser Gly Leu 675 680 685Leu His Ser Ile Thr Leu Leu Ala His Leu Thr Ser Asp Ala Ile Ala 690 695700 His Gly Ala Thr Thr Cys Leu Arg Ala Pro Pro Glu Pro Ala Asp Ser 705710 715 720 Leu Thr Glu Ala Cys Lys Gln Tyr Gly Arg Glu Thr Leu Ala TyrLeu 725 730 735 Ala Ser Leu Glu Glu Glu Gly Ser Leu Glu Asn Ala Asp SerThr Ala 740 745 750 Met Arg Asn Cys Leu Ser Lys Ile Lys Ala Ile Gly GluGlu Leu Leu 755 760 765 Pro Arg Gly Leu Asp Ile Lys Gln Glu Glu Leu GlyAsp Leu Val Asp 770 775 780 Lys Glu Met Ala Ala Thr Ser Ala Ala Ile GluThr Ala Thr Ala Arg 785 790 795 800 Ile Glu Glu Met Leu Ser Lys Ser ArgAla Gly Asp Thr Gly Val Lys 805 810 815 Leu Glu Val Asn Glu Arg Ile LeuGly Cys Cys Thr Ser Leu Met Gln 820 825 830 Ala Ile Gln Val Leu Ile ValAla Ser Lys Asp Leu Gln Arg Glu Ile 835 840 845 Val Glu Ser Gly Arg GlyThr Ala Ser Pro Lys Glu Phe Tyr Ala Lys 850 855 860 Asn Ser Arg Trp ThrGlu Gly Leu Ile Ser Ala Ser Lys Ala Val Gly 865 870 875 880 Trp Gly AlaThr Val Met Val Asp Ala Ala Asp Leu Val Val Gln Gly 885 890 895 Arg GlyLys Phe Glu Glu Leu Met Val Cys Ser His Glu Ile Ala Ala 900 905 910 SerThr Ala Gln Leu Val Ala Ala Ser Lys Val Lys Ala Asp Lys Asp 915 920 925Ser Pro Asn Leu Ala Gln Leu Gln Gln Ala Ser Arg Gly Val Asn Gln 930 935940 Ala Thr Ala Gly Val Val Ala Ser Thr Ile Ser Gly Lys Ser Gln Ile 945950 955 960 Glu Glu Thr Asp Asn Met Asp Phe Ser Ser Met Thr Leu Thr GlnIle 965 970 975 Lys Arg Gln Glu Met Asp Ser Gln Val Arg Val Leu Glu LeuGlu Asn 980 985 990 Glu Leu Gln Lys Glu Arg Gln Lys Leu Gly Glu Leu ArgLys Lys His 995 1000 1005 Tyr Glu Leu Ala Gly Val Ala Glu Gly Trp GluGlu Gly Thr Glu 1010 1015 1020 Ala Ser Pro Pro Thr Leu Gln Glu Val ValThr Glu Lys Glu Ser 1025 1030 1035 Gln Thr Asn Thr Pro Tyr Val Ser ValAsn Pro Cys Tyr Leu Ser 1040 1045 1050 Arg Val Cys Tyr Phe Pro Ser HisArg Pro Asn Pro Trp Ser Pro 1055 1060 1065 Arg Gly Ser His Thr Thr AlaIle Thr Gln Cys Arg Gly His Ala 1070 1075 1080 His Phe Gln Arg Leu ProPro Arg His Pro Phe Cys Leu Asp Pro 1085 1090 1095 Trp Ile Ser Thr AlaSer Tyr Gly Gly Trp Leu Gly Phe Leu Val 1100 1105 1110 Leu Phe Phe PhePhe Lys Phe His Ser His Ser Gln Leu Ser Gln 1115 1120 1125 Arg Ala HisPro Trp Gly Val Ser Arg Ala Pro Gln Leu Trp Leu 1130 1135 1140 Gln ArgTrp Cys Cys Pro Gly Leu Ser Val Leu His Leu Arg Leu 1145 1150 1155 HisThr Asp Gln Val Leu Ala His Pro Val His Ala Pro Gly Ser 1160 1165 1170Gly Gly Ala Ala Glu Gln Leu Ser Ser Lys Ser Arg Arg Arg Val 1175 11801185 Ser Ala Phe Pro Ser Ser Ile Pro Ala Glu Ser Leu Cys Pro Pro 11901195 1200 Leu Gln Gly Arg Arg Gln Gln Lys Glu Gly Gln Glu Gly Ser His1205 1210 1215 Ser Pro Val Pro Val Thr Arg Leu Lys Asn Leu Ile Thr CysLeu 1220 1225 1230 Asn Gly Ala Gly Glu Ile Asn Asn Thr Thr Ser Leu ProGlu Thr 1235 1240 1245 Val Arg Glu Trp Ser Leu Ser Ser Gly Pro Ser ProLeu Ala Gln 1250 1255 1260 Arg Arg Ser Val Gly Val Ile Pro Asn Ser PheLeu Gln Thr Ser 1265 1270 1275 Ala Leu Ala Ser Ser Ile Gly Arg Ser PheHis Leu Leu Arg Asn 1280 1285 1290 Gln Thr Arg Lys Ile Arg Cys Asn CysSer His Gln Gly Arg Thr 1295 1300 1305 Leu Tyr Leu Val Cys Tyr Pro TyrLeu Leu Leu Thr Ser Leu Lys 1310 1315 1320 Gln Gln Gln Pro Thr Lys ArgCys Leu Glu Gln Ser Glu Leu Gln 1325 1330 1335 Val Leu Gln Ser Ser SerPhe Cys Pro Ala Thr Ser Ala Phe Lys 1340 1345 1350 Asn Gln Lys Lys GlyGln Gly Ala Gly Leu Leu Leu Thr Trp Ile 1355 1360 1365 Pro Lys Gln GlyAsp His Leu Glu Leu Leu Gly Gln Arg Lys Glu 1370 1375 1380 Arg Thr GluPro Ala Ala Pro Thr Pro Phe Ser His Met Pro Gln 1385 1390 1395 Ala LeuAla Ala Leu Trp Thr Gly Gly Gln Arg Ala His Glu Gln 1400 1405 1410 LeuAla Arg Asp Gly Gln Pro Asn Ser Thr Phe Pro Leu Leu Asp 1415 1420 1425Gly Pro Gln His Leu Ser Asp Leu Leu Ile Leu Gly Lys Gln Arg 1430 14351440 Leu Pro Ser Leu Ser Ile Ala Thr His Trp Trp Pro Ser Ser Thr 14451450 1455 Ser Glu Phe Leu Gln Pro Gly Arg Pro Leu Glu His Ala Xaa Glu1460 1465 1470 Gly Pro 1475 3 3911 DNA Homo sapiens 3 gttaacagtggagatgtttg actacctgga gtgtgaactc aacctcttcc aaacagtatt 60 caactccctggacatgtccc gctctgtgtc cgtgacggca gcagggcagt gccgcctcgc 120 cccgctgatccaggtcatct tggactgcag ccacctttat gactacactg tcaagcttct 180 cttcaaactccactcctgcc tcccagctga caccctgcaa ggccaccggg accgcttcat 240 ggagcagtttacaaagttga aagatctgtt ctaccgctcc agcaacctgc agtacttcaa 300 gcggctcattcagatccccc agctgcctga gaacccaccc aacttcctgc gagcctcagc 360 cctgtcagaacatatcagcc ctgtggtggt gatccctgca gaggcctcat cccccgacag 420 cgagccagtcctagagaagg atgacctcat ggacatggat gcctctcagc agaatttatt 480 tgacaacaagtttgatgaca tctttggcag ttcattcagc agtgatccct tcaatttcaa 540 cagtcaaaatggtgtgaaca aggatgagaa ggaccactta attgagcgac tatacagaga 600 gatcagtggattgaaggcac agctagaaaa catgaagact gagagccagc gggttgtgct 660 gcagctgaagggccacgtca gcgagctgga agcagatctg gccgagcagc agcacctgcg 720 gcagcaggcggccgacgact gtgaattcct gcgggcagaa ctggacgagc tcaggaggca 780 gcgggaggacaccgagaagg ctcagcggag cctgtctgag atagaaagga aagctcaagc 840 caatgaacagcgatatagca agctaaagga gaagtacagc gagctggttc agaaccacgc 900 tgacctgctgcggaagaatg cagaggtgac caaacaggtg tccatggcca gacaagccca 960 ggtagatttggaacgagaga aaaaagagct ggaggattcg ttggagcgca tcagtgacca 1020 gggccagcggaagactcaag aacagctgga agttctagag agcttgaagc aggaacttgc 1080 cacaagccaacgggagcttc aggttctgca aggcagcctg gaaacttctg cccagtcaga 1140 agcaaactgggcagccgagt tcgccgagct agagaaggag cgggacagcc tggtgagtgg 1200 cgcagctcatagggaggagg aattatctgc tcttcggaaa gaactgcagg acactcagct 1260 caaactggccagcacagagg aatctatgtg ccagcttgcc aaagaccaac gaaaaatgct 1320 tctggtggggtccaggaagg ctgcggagca ggtgatacaa gacgccctga accagcttga 1380 agaacctcctctcatcagct gcgctgggtc tgcagatcac ctcctctcca cggtcacatc 1440 catttccagctgcatcgagc aactggagaa aagctggagc cagtatctgg cctgcccaga 1500 agacatcagtggacttctcc attccataac cctgctggcc cacttgacca gcgacgccat 1560 tgctcatggtgccaccacct gcctcagagc cccacctgag cctgccgact cactgaccga 1620 ggcctgtaagcagtatggca gggaaaccct cgcctacctg gcctccctgg aggaagaggg 1680 aagccttgagaatgccgaca gcacagccat gaggaactgc ctgagcaaga tcaaggccat 1740 cggcgaggagctcctgccca ggggactgga catcaagcag gaggagctgg gggacctggt 1800 ggacaaggagatggcggcca cttcagctgc tattgaaact gccacggcca gaatagagga 1860 gatgctcagcaaatcccgag caggagacac aggagtcaaa ttggaggtga atgaaaggat 1920 ccttggttgctgtaccagcc tcatgcaagc tattcaggtg ctcatcgtgg cctctaagga 1980 cctccagagagagattgtgg agagcggcag gggtacagca tcccctaaag agttttatgc 2040 caagaactctcgatggacag aaggacttat ctcagcctcc aaggctgtgg gctggggagc 2100 cactgtcatggtggatgcag ctgatctggt ggtacaaggc agagggaaat ttgaggagct 2160 aatggtgtgttctcatgaaa ttgctgctag cacagcccag cttgtggctg catccaaggt 2220 gaaagctgataaggacagcc ccaacctagc ccagctgcag caggcctctc ggggagtgaa 2280 ccaggccactgccggcgttg tggcctcaac catttccggc aaatcacaga tcgaagagac 2340 agacaacatggacttctcaa gcatgacgct gacacagatc aaacgccaag agatggattc 2400 tcaggttagggtgctagagc tagaaaatga attgcagaag gagcgtcaaa aactgggaga 2460 gcttcggaaaaagcactacg agcttgctgg tgttgctgag ggctgggaag aaggaacaga 2520 ggcatctccacctacactgc aagaagtggt aaccgaaaaa gaatagagcc aaaccaacac 2580 cccatatgtcagtgtaaatc cttgttacct atctcgtgtg tgttatttcc ccagccacag 2640 gccaaatccttggagtccca ggggcagcca caccactgcc attacccagt gccgaggaca 2700 tgcatgacacttccaaagac tccctccata gcgacaccct ttctgtttgg acccatggat 2760 ttccactgcttcttatggtg gttggttggg ttttttggtt ttgttttttt tttttaagtt 2820 tcactcacatagccaactct cccaaagggc acacccctgg ggctgagtct ccagggcccc 2880 ccaactgtggtagctccagc gatggtgctg cccaggcctc tcggtgctcc atctccgcct 2940 ccacactgaccaagtgctgg cccacccagt ccatgctcca gggtcaggcg gagctgctga 3000 gtgacagctttcctcaaaaa gcagaaggag agtgagtgcc tttccctcct aaagctgaat 3060 cccggcggaaagcctctgtc cgcctttaca agggagaaga caacagaaag agggacaaga 3120 gggttcacacagcccagttc ccgtgacgag gctcaaaaac ttgatcacat gcttgaatgg 3180 agctggtgagatcaacaaca ctacttccct gccggaatga actgtccgtg aatggtctct 3240 gtcaagcgggccgtctccct tggcccagag acggagtgtg ggagtgattc ccaactcctt 3300 tctgcagacgtctgccttgg catcctcttg aataggaaga tcgttccacc ttctacgcaa 3360 ttgacaaacccggaagatca gatgcaattg ctcccatcag ggaagaaccc tatacttggt 3420 ttgctacccttagtatttat tactaacctc ccttaagcag caacagccta caaagagatg 3480 cttggagcaatcagaacttc aggtgtgact ctagcaaagc tcatctttct gcccggctac 3540 atcagccttcaagaatcaga agaaaggcca aggtgctgga ctgttactga cttggatccc 3600 aaagcaaggagatcatttgg agctcttggg tcagagaaaa tgagaaagga cagagccagc 3660 ggctccaactcctttcagcc acatgcccca ggctctcgct gccctgtgga caggatgagg 3720 acagagggcacatgaacagc ttgccaggga tgggcagccc aacagcactt ttcctcttct 3780 agatggaccccagcatttaa gtgaccttct gatcttggga aaacagcgtc ttccttcttt 3840 atctatagcaactcattggt ggtagccatc aagcacttcg gaattcctgc agcccgggcg 3900 gccgctcgag c3911 4 1283 PRT Homo sapiens 4 Met Phe Asp Tyr Leu Glu Cys Glu Leu AsnLeu Phe Gln Thr Val Phe 1 5 10 15 Asn Ser Leu Asp Met Ser Arg Ser ValSer Val Thr Ala Ala Gly Gln 20 25 30 Cys Arg Leu Ala Pro Leu Ile Gln ValIle Leu Asp Cys Ser His Leu 35 40 45 Tyr Asp Tyr Thr Val Lys Leu Leu PheLys Leu His Ser Cys Leu Pro 50 55 60 Ala Asp Thr Leu Gln Gly His Arg AspArg Phe Met Glu Gln Phe Thr 65 70 75 80 Lys Leu Lys Asp Leu Phe Tyr ArgSer Ser Asn Leu Gln Tyr Phe Lys 85 90 95 Arg Leu Ile Gln Ile Pro Gln LeuPro Glu Asn Pro Pro Asn Phe Leu 100 105 110 Arg Ala Ser Ala Leu Ser GluHis Ile Ser Pro Val Val Val Ile Pro 115 120 125 Ala Glu Ala Ser Ser ProAsp Ser Glu Pro Val Leu Glu Lys Asp Asp 130 135 140 Leu Met Asp Met AspAla Ser Gln Gln Asn Leu Phe Asp Asn Lys Phe 145 150 155 160 Asp Asp IlePhe Gly Ser Ser Phe Ser Ser Asp Pro Phe Asn Phe Asn 165 170 175 Ser GlnAsn Gly Val Asn Lys Asp Glu Lys Asp His Leu Ile Glu Arg 180 185 190 LeuTyr Arg Glu Ile Ser Gly Leu Lys Ala Gln Leu Glu Asn Met Lys 195 200 205Thr Glu Ser Gln Arg Val Val Leu Gln Leu Lys Gly His Val Ser Glu 210 215220 Leu Glu Ala Asp Leu Ala Glu Gln Gln His Leu Arg Gln Gln Ala Ala 225230 235 240 Asp Asp Cys Glu Phe Leu Arg Ala Glu Leu Asp Glu Leu Arg ArgGln 245 250 255 Arg Glu Asp Thr Glu Lys Ala Gln Arg Ser Leu Ser Glu IleGlu Arg 260 265 270 Lys Ala Gln Ala Asn Glu Gln Arg Tyr Ser Lys Leu LysGlu Lys Tyr 275 280 285 Ser Glu Leu Val Gln Asn His Ala Asp Leu Leu ArgLys Asn Ala Glu 290 295 300 Val Thr Lys Gln Val Ser Met Ala Arg Gln AlaGln Val Asp Leu Glu 305 310 315 320 Arg Glu Lys Lys Glu Leu Glu Asp SerLeu Glu Arg Ile Ser Asp Gln 325 330 335 Gly Gln Arg Lys Thr Gln Glu GlnLeu Glu Val Leu Glu Ser Leu Lys 340 345 350 Gln Glu Leu Ala Thr Ser GlnArg Glu Leu Gln Val Leu Gln Gly Ser 355 360 365 Leu Glu Thr Ser Ala GlnSer Glu Ala Asn Trp Ala Ala Glu Phe Ala 370 375 380 Glu Leu Glu Lys GluArg Asp Ser Leu Val Ser Gly Ala Ala His Arg 385 390 395 400 Glu Glu GluLeu Ser Ala Leu Arg Lys Glu Leu Gln Asp Thr Gln Leu 405 410 415 Lys LeuAla Ser Thr Glu Glu Ser Met Cys Gln Leu Ala Lys Asp Gln 420 425 430 ArgLys Met Leu Leu Val Gly Ser Arg Lys Ala Ala Glu Gln Val Ile 435 440 445Gln Asp Ala Leu Asn Gln Leu Glu Glu Pro Pro Leu Ile Ser Cys Ala 450 455460 Gly Ser Ala Asp His Leu Leu Ser Thr Val Thr Ser Ile Ser Ser Cys 465470 475 480 Ile Glu Gln Leu Glu Lys Ser Trp Ser Gln Tyr Leu Ala Cys ProGlu 485 490 495 Asp Ile Ser Gly Leu Leu His Ser Ile Thr Leu Leu Ala HisLeu Thr 500 505 510 Ser Asp Ala Ile Ala His Gly Ala Thr Thr Cys Leu ArgAla Pro Pro 515 520 525 Glu Pro Ala Asp Ser Leu Thr Glu Ala Cys Lys GlnTyr Gly Arg Glu 530 535 540 Thr Leu Ala Tyr Leu Ala Ser Leu Glu Glu GluGly Ser Leu Glu Asn 545 550 555 560 Ala Asp Ser Thr Ala Met Arg Asn CysLeu Ser Lys Ile Lys Ala Ile 565 570 575 Gly Glu Glu Leu Leu Pro Arg GlyLeu Asp Ile Lys Gln Glu Glu Leu 580 585 590 Gly Asp Leu Val Asp Lys GluMet Ala Ala Thr Ser Ala Ala Ile Glu 595 600 605 Thr Ala Thr Ala Arg IleGlu Glu Met Leu Ser Lys Ser Arg Ala Gly 610 615 620 Asp Thr Gly Val LysLeu Glu Val Asn Glu Arg Ile Leu Gly Cys Cys 625 630 635 640 Thr Ser LeuMet Gln Ala Ile Gln Val Leu Ile Val Ala Ser Lys Asp 645 650 655 Leu GlnArg Glu Ile Val Glu Ser Gly Arg Gly Thr Ala Ser Pro Lys 660 665 670 GluPhe Tyr Ala Lys Asn Ser Arg Trp Thr Glu Gly Leu Ile Ser Ala 675 680 685Ser Lys Ala Val Gly Trp Gly Ala Thr Val Met Val Asp Ala Ala Asp 690 695700 Leu Val Val Gln Gly Arg Gly Lys Phe Glu Glu Leu Met Val Cys Ser 705710 715 720 His Glu Ile Ala Ala Ser Thr Ala Gln Leu Val Ala Ala Ser LysVal 725 730 735 Lys Ala Asp Lys Asp Ser Pro Asn Leu Ala Gln Leu Gln GlnAla Ser 740 745 750 Arg Gly Val Asn Gln Ala Thr Ala Gly Val Val Ala SerThr Ile Ser 755 760 765 Gly Lys Ser Gln Ile Glu Glu Thr Asp Asn Met AspPhe Ser Ser Met 770 775 780 Thr Leu Thr Gln Ile Lys Arg Gln Glu Met AspSer Gln Val Arg Val 785 790 795 800 Leu Glu Leu Glu Asn Glu Leu Gln LysGlu Arg Gln Lys Leu Gly Glu 805 810 815 Leu Arg Lys Lys His Tyr Glu LeuAla Gly Val Ala Glu Gly Trp Glu 820 825 830 Glu Gly Thr Glu Ala Ser ProPro Thr Leu Gln Glu Val Val Thr Glu 835 840 845 Lys Glu Ser Gln Thr AsnThr Pro Tyr Val Ser Val Asn Pro Cys Tyr 850 855 860 Leu Ser Arg Val CysTyr Phe Pro Ser His Arg Pro Asn Pro Trp Ser 865 870 875 880 Pro Arg GlySer His Thr Thr Ala Ile Thr Gln Cys Arg Gly His Ala 885 890 895 His PheGln Arg Leu Pro Pro Arg His Pro Phe Cys Leu Asp Pro Trp 900 905 910 IleSer Thr Ala Ser Tyr Gly Gly Trp Leu Gly Phe Leu Val Leu Phe 915 920 925Phe Phe Phe Lys Phe His Ser His Ser Gln Leu Ser Gln Arg Ala His 930 935940 Pro Trp Gly Val Ser Arg Ala Pro Gln Leu Trp Leu Gln Arg Trp Cys 945950 955 960 Cys Pro Gly Leu Ser Val Leu His Leu Arg Leu His Thr Asp GlnVal 965 970 975 Leu Ala His Pro Val His Ala Pro Gly Ser Gly Gly Ala AlaGlu Gln 980 985 990 Leu Ser Ser Lys Ser Arg Arg Arg Val Ser Ala Phe ProSer Ser Ile 995 1000 1005 Pro Ala Glu Ser Leu Cys Pro Pro Leu Gln GlyArg Arg Gln Gln 1010 1015 1020 Lys Glu Gly Gln Glu Gly Ser His Ser ProVal Pro Val Thr Arg 1025 1030 1035 Leu Lys Asn Leu Ile Thr Cys Leu AsnGly Ala Gly Glu Ile Asn 1040 1045 1050 Asn Thr Thr Ser Leu Pro Glu ThrVal Arg Glu Trp Ser Leu Ser 1055 1060 1065 Ser Gly Pro Ser Pro Leu AlaGln Arg Arg Ser Val Gly Val Ile 1070 1075 1080 Pro Asn Ser Phe Leu GlnThr Ser Ala Leu Ala Ser Ser Ile Gly 1085 1090 1095 Arg Ser Phe His LeuLeu Arg Asn Gln Thr Arg Lys Ile Arg Cys 1100 1105 1110 Asn Cys Ser HisGln Gly Arg Thr Leu Tyr Leu Val Cys Tyr Pro 1115 1120 1125 Tyr Leu LeuLeu Thr Ser Leu Lys Gln Gln Gln Pro Thr Lys Arg 1130 1135 1140 Cys LeuGlu Gln Ser Glu Leu Gln Val Leu Gln Ser Ser Ser Phe 1145 1150 1155 CysPro Ala Thr Ser Ala Phe Lys Asn Gln Lys Lys Gly Gln Gly 1160 1165 1170Ala Gly Leu Leu Leu Thr Trp Ile Pro Lys Gln Gly Asp His Leu 1175 11801185 Glu Leu Leu Gly Gln Arg Lys Glu Arg Thr Glu Pro Ala Ala Pro 11901195 1200 Thr Pro Phe Ser His Met Pro Gln Ala Leu Ala Ala Leu Trp Thr1205 1210 1215 Gly Gly Gln Arg Ala His Glu Gln Leu Ala Arg Asp Gly GlnPro 1220 1225 1230 Asn Ser Thr Phe Pro Leu Leu Asp Gly Pro Gln His LeuSer Asp 1235 1240 1245 Leu Leu Ile Leu Gly Lys Gln Arg Leu Pro Ser LeuSer Ile Ala 1250 1255 1260 Thr His Trp Trp Pro Ser Ser Thr Ser Glu PheLeu Gln Pro Gly 1265 1270 1275 Arg Pro Leu Glu His 1280 5 21 DNA Homosapiens 5 gctgtaaagg aaaaacacgc c 21 6 404 DNA Homo sapiens 6 gggccgagccagcggagggg ctcctgaagg ggcgggggcg ggcggggaag ccgttcggcg 60 aggggcggggtctctggaag actggcagaa ctcacagcca atggcaggcg ggagccgtcc 120 cgttagcgccggatccccgc gggtagggcg gggcgggcgg cgccgtgggg atcccggggc 180 agccgagggcccctgactcg gctcctcgcg gcgacatgga tcggatggcc agctccatga 240 agcaggtgcccaacccactg cccaaggtgc tgagccggcg cggggtcggc gctgggctgg 300 aggcggcggagcgcgagagc ttcgagcgga ctcaggttca gactgtcagc atcaataagg 360 ccattaatacgcaggaaagt ggctgtaaag gaaaaacatg ccag 404

We claim:
 1. An antibody that specifically binds to HIP1 but does notspecifically bind to the normal epithelium of prostate or colon.
 2. Theantibody of claim 1, wherein said antibody binds to the cancerousepithelium of colon or prostate but does not bind to the normalepithelium of prostate or colon.
 3. The antibody of claim 1, whereinsaid antibody is a monoclonal antibody.
 4. The monoclonal antibody ofclaim 3, wherein said antibody has substantially the same properties asantibodies secreted by a hybridoma selected from the group consisting ofthose deposited as ATCC numbers pending.
 5. The monoclonal antibody ofclaim 4, wherein said antibody is secreted by a hybridoma with ATCCdeposit number pending.
 6. The monoclonal antibody of claim 4, whereinsaid antibody is secreted by a hybridoma with ATCC deposit numberpending.
 7. The monoclonal antibody of claim 4, wherein said antibodyspecifically binds to HIP1 protein with low background binding.
 8. Themonoclonal antibody of claim 4, wherein said antibody binds to human andmouse HIP1.
 9. A method for detecting cancer, comprising: a) providing asample from a subject suspected of having cancer; and b) detecting thepresence or absence of HIP1 in said sample.
 10. The method of claim 9,wherein the presence of HIP1 in said sample is indicative of cancer insaid subject.
 11. The method of claim 9, wherein said cancer is selectedfrom the group consisting of prostate cancer and colon cancer.
 12. Themethod of claim 9, wherein said sample is a tumor sample.
 13. The methodof claim 9, wherein said sample is a tissue sample.
 14. The method ofclaim 13, wherein said tissue sample is selected from the groupconsisting of prostate tissue and colon tissue.
 15. The method of claim9, wherein said sample is selected from the group consisting of serum,plasma, blood, and urine.
 16. The method of claim 8, wherein saiddetecting HIP1 comprises detecting the presence of HIP1 mRNA.
 17. Themethod of claim 16, wherein said detecting the presence of HIP1 mRNAcomprises exposing said HIP1 mRNA to a nucleic acid probe complementaryto at least a portion of said HIP1 mRNA.
 18. The method of claim 17,wherein said detecting the presence of HIP1 mRNA comprises a detectionassay selected from the group consisting of a Northern blot, in situhybridization, reverse-transcriptase polymerase chain reaction, andmicroarray analysis.
 19. The method of claim 9, wherein said detectingthe presence of HIP1 comprises detecting the presence of a HIP1polypeptide.
 20. The method of claim 17, wherein said detecting thepresence of a HIP1 polypeptide comprises exposing said HIP1 polypeptideto an antibody that specifically binds to HIP1 but does not specificallybind to the normal epithelium of prostate or colon and detecting thebinding of said antibody to said HIP1 polypeptide.
 21. The method ofclaim 20, wherein said antibody is a monoclonal antibody.
 22. The methodof claim 21, wherein said monoclonal antibody has substantially the sameproperties as monoclonal antibodies secreted by a hybridoma selectedfrom the group consisting of those deposited as ATCC numbers pending.23. The method of claim 9, wherein said method further comprises step c)providing a prognosis to said subject.
 24. A method for characterizingtissue in a subject, comprising: a) providing a tissue sample from asubject, wherein said tissue is selected from the group consisting ofcolon and prostate tissue; and b) detecting the presence or absence ofHIP1 in said sample, thereby characterizing said tissue sample.
 25. Themethod of claim 24, wherein said tissue is tumor tissue.
 26. The methodof claim 24, wherein said tissue is biopsy tissue.
 27. The method ofclaim 24, wherein said detecting HIP1 comprises detecting the presenceof HIP1 mRNA.
 28. The method of claim 27, wherein said detecting thepresence of HIP1 mRNA comprises exposing said HIP1 mRNA to a nucleicacid probe complementary to at least a portion of said HIP1 mRNA. 29.The method of claim 28, wherein said detecting the presence of HIP1 mRNAcomprises a detection assay selected from the group consisting of aNorthern blot, in situ hybridization, reverse-transcriptase polymerasechain reaction, and microarray analysis.
 30. The method of claim 24,wherein said detecting the presence of HIP1 comprises detecting thepresence of a HIP1 polypeptide.
 31. The method of claim 30, wherein saiddetecting the presence of a HIP1 polypeptide comprises exposing saidHIP1 polypeptide to an antibody that specifically binds to HIP1 but doesnot specifically bind to the normal epithelium of prostate or colon anddetecting the binding of said antibody to said HIP1 polypeptide.
 32. Themethod of claim 31, wherein said antibody is a monoclonal antibody. 33.The method of claim 32, wherein said monoclonal antibody hassubstantially the same properties as monoclonal antibodies secreted by ahybridoma selected from the group consisting of those deposited as ATCCnumbers pending.
 34. The method of claim 24, wherein said tissue sampleis a post-surgical prostate tumor tissue sample and said method furthercomprises the step of c) identifying a risk of prostate specific antigenfailure based on said detecting the presence of HIP1.
 35. The method ofclaim 24, wherein said tissue sample is prostate tumor tissue and saidcharacterizing comprises identifying a stage of prostate cancer in saidprostate tumor tissue.
 36. The method of claim 35, wherein said stage isselected from the group consisting of high-grade prostaticintraepithelial neoplasia, benign prostatic hyperplasia, prostatecarcinoma, and metastatic prostate carcinoma.
 37. The method of claim24, wherein said tissue sample is prostate tumor tissue and said methodfurther comprises the step of c) identifying the risk of said tumormetastasizing based on said detecting the presence of HIP1.
 38. Themethod of claim 24, wherein said tissue sample is post-surgical prostatetumor tissue and said method further comprises the step of c)identifying the risk of said tumor recurring based on said detecting thepresence of HIP1.
 39. A kit for characterizing cancer in a subject,comprising: a) a reagent that specifically detects the presence ofabsence of expression of HIP1; and b) instructions for using said kitfor characterizing cancer in said subject.
 40. The kit of claim 39,wherein said reagent comprises an antibody that specifically binds toHIP1 but does not specifically bind to the normal epithelium of prostateor colon.
 41. The kit of claim 40, wherein said antibody is a monoclonalantibody.
 42. The kit of claim 41, wherein said monoclonal antibody hassubstantially the same properties as monoclonal antibodies secreted by ahybridoma selected from the group consisting of those deposited as ATCCnumbers pending.
 43. The kit of claim 39, wherein said reagent comprisesa nucleic acid probe that specifically binds to a HIP1 mRNA.
 44. The kitof claim 39, wherein said instructions comprise instructions required bythe United States Food and Drug Administration for use in in vitrodiagnostic products.
 45. A method of screening compounds, comprising: a)providing i) an cell sample comprising cancerous epithelial cells; andii) one or more test compounds; and b) contacting said sample with saidtest compound; and c) detecting a change in HIP1 expression in saidsample in the presence of said test compound relative to the absence ofsaid test compound.
 46. The method of claim 45, wherein said contactingsaid sample with said test compound results in death of said cancerousepithelial cells.
 47. The method of claim 45, wherein said contactingsaid sample with said test compound results in impaired proliferation ofsaid cancerous epithelial cells.
 48. The method of claim 45, whereinsaid epithelial cell sample is selected from the group consisting ofprostate cancer cells and colon cancer cells.
 49. The method of claim45, wherein said detecting comprises detecting HIP1 mRNA.
 50. The methodof claim 45, wherein said detecting comprises detecting HIP1polypeptide.
 51. The method of claim 45, wherein said cell is in vitro.52. The method of claim 45, wherein said cell is in vivo.
 53. The methodof claim 45, wherein said test compound comprises an antisense compound.54. The method of claim 45, wherein said test compound comprises a drug.55. The method of claim 54, wherein said drug is an antibody.
 56. Themethod of claim 54, wherein said drug specifically binds to HIP1.
 57. Amethod of screening compounds, comprising: a) providing i) a first cellsample comprising cells expressing wild-type HIP1; ii) a second cellsample comprising cells, wherein said cells do not express HIP1; iii)one or more test compounds; and b) contacting said first and secondssamples with said test compound; and c) detecting a decrease inviability in said first sample relative to said second sample.
 58. Themethod of claim 57, wherein said decrease in viability is due toprogrammed cell death.
 59. The method of claim 57, wherein said firstand second cell samples comprise embryonic fibroblast cells.
 60. Themethod of claim 58, wherein first cell sample comprises embryonicfibroblast cells derived from wild-type mice.
 61. The method of claim59, wherein said second cell sample comprises embryonic fibroblast cellsderived from HIP1 knockout mice.
 62. The method of claim 57, whereinsaid first and second cell samples comprise first and second humancancer cell lines.
 63. The method of claim 62, wherein said second humancancer cell line is colo205 cells.
 64. The method of claim 57, whereinsaid test compound comprises a library of test compounds.
 65. The methodof claim 57, wherein said test compound comprises a lipid analogue. 66.The method of claim 57, wherein said test compound binds to HIP1. 67.The method of claim 66, wherein said test compound binds to the ENTHdomain of HIP1.
 68. A composition comprising a mutant HIP1 nucleic acidsequence, said sequence lacking the ENTH domain.
 69. The composition ofclaim 68, wherein said nucleic acid sequence comprises SEQ ID NO:
 3. 70.A composition comprising a polypeptide encoded by the nucleic acidsequence of claim
 68. 71. A composition comprising a mutant HIP1polypeptide, wherein said polypeptide induces cell death when expressedin a cell.
 72. The composition of claim 71, wherein said mutant HIP1polypeptide is lacking a ENTH domain.
 73. The composition of claim 72,wherein said HIP1 polypeptide comprises SEQ ID NO:
 4. 74. A nucleic acidsequence encoding the polypeptide of claim
 71. 75. A non-humantransgenic animal lacking a functional HIP1 gene.
 76. The non-humantransgenic animal of claim 75, wherein said animal is a mouse.
 77. Thenon-human transgenic animal of claim 75, wherein said animal comprises aknock-out of the HIP1 gene.
 78. The non-human transgenic animal of claim76, wherein said knock-out of the HIP1 gene is a conditional knock-out.79. The non-human transgenic animal of claim 75, wherein said animalcomprises a knock-in of the HIP1 gene.
 80. A composition comprising adrug, wherein said drug binds to wild type HIP1 but not a HIP1 ENTHdeletion mutant, and wherein said drug inhibits HIP1 biologicalactivity.
 81. The composition of claim 80, wherein said drug binds tothe ENTH domain of HIP1.
 82. The composition of claim 81, wherein saiddrug is a lipid analogue.
 83. The composition of claim 82, wherein saidlipid analogue is a phosphoinositide mimetic.